THE PREPARATION OF CHEMICALLY-MODIFIED CYS-TRNA~~' AND STUDIES ON ITS POSSIBLE USE FOR INCORPORATING NON-NATURAL AMIN0 AClDS INTO PROTEINS
Linda Lien
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Chemistry
University of Toronto
O Copyright by Linda Lien 2001 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. nie WelEngton Ottawa ON K1A ON4 Ottawa ON KIA ON4 Canada Canada
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Doctor of Philosophy 2001 Linda Lien Graduate Department of Chemistry University of Toronto
Abstract
Biosynthetic incorporation of fluorophores into proteins can be accomplished by supplying fluorescently-tagged aminoacyl-tRNA (aa-tRNA) to the cellular protein synthesis machiner=. A key step in this process is the formation of a ternary complex consisting of an elongation factor (EF-TU in bacteria), GTP, and the aminoacyl-tRNA. In an effort to explore the range of side-chain structure that can be tolerated by EF-TU.we prepared C~S-~RNA~~~ and reacted the cysteinyl side-chain with a structurally diverse set of fluorescent compounds. AI1 modified aa-tRNA tested were found to bind to EF-Tu-GTP and binding was specific as dernonstrated by the lack of an interaction with immobilized EF-TU-GDP. These results are compared with the predictions of rnodelling based on the recently reported X-ray structure of the C~S-~RNA~~~-EF-
TU-GDPNP cornplex. It appears that, except perhaps for certain modifications very close to the backbone of the amino acid, a very wide range of side-chain structures can be tolerated by EF-TU.
We show that reverse-phase high performance liquid chromatography using a C8 column can be ernployed to purify fluorescently-labelled C~S-~RNA~~' from a crude mixture which also contains unaminoacylated ~RNA'Y', unlabelled
C~S-~RNA~~~,and disulphide-linked C~S-~RNA~~~.
Towards expanding the range of methods available for site-specific protein labelling, we proposed a technique which is based on the use of chernically- modified ~~stein~l-t~~~~~~.The synthetic tRNA is synthesized via a combination of enzymatic aminoacylation and chemical modification of the cysteinyl side- chain. When it is added to a rabbit reticulocyte lysate in vitro coupled transcription/translation system that has been programmed with the gene of interest, the non-standard amino acid should be incorporated into the target protein at positions that correspond to cysteine. We attempted to label firefly luciferase with Ruorescein and a biotin derivative using fluorescein- and biotin- labelled C~S-~RNA~~~,respectively. Acknowledgments
The first and foremost person for me to acknowledge is my supervisor
(Prof. Woolley). 1 would Iike to thank him for his teachings, guidance,
encouragement, endless patience, and belief in me. He was extremely helpful
but at the same time gave me the opportunity to develop into an independent
researcher. I will be eternally grateful.
My CO-workers,in no particular order, were Vitali, Darcy, Janet, Tyler.
Jack, Christine, Dom, Ananda, and Andrew. I would like to acknowledge them for the helpful discussions in chemistry and biochernistry. But most importantly, I would like to thank them for their friendship. The friendships and lunches at
Lucky Dragon are some of the things I will miss.
I would also like to thank MRC (Medical Research Council of Canada) for providing the funding for my project. I am grateful to the University of Toronto. my supervisor, and NSERC (Natural Science and Engineering Research Council of Canada) for providing my salary.
For my Mom and Dad, the two most courageous people I know, 1 thank them for their patience and for putting up with me. They were responsible for teaching me the value of hard work and dedication. I wiil be eternally grateful to them for al1 the sacrifices they made so as to give me the opportunity to achieve my dreams.
1 would also like to thank the other members of my farnily (Heng, Cindy,
Quang, Dad MacKay, Mom MacKay, Scott, Mary-Janet, and Richard) for their love, support, and patience. Finally, I would like to acknowledge my husband and best friend Bruce. I would like to thank him for his love, support, cornfort, and patience. Without him, this thesis would not have been possible. To Bruce, and to my parents Table of Contents
Abstract
Acknowledgements iv
Table of Contents vii
List of Tables XV
List of Schemes
List of Figures
List of Appendices
List of AbbreviationslDefinitions
Chapter 1 Interaction of Fluorescently-Labelled C~S-~RNA~~~with EF-Tu-GTP 1
1 1 Introduction
1.1.1 Significance of EF-Tu in biosynthetic incorporation of fluorescent non-natural amino acids into proteins by non-sense suppression
1 .l .7.7 General overview of protein synthesis in E. coii with emphasis on the role of EF-Tu
1.1 -2 EF-Tu background
1.1 -2.1 Function of EF-Tu
1.1 -2.2 Structure of GDP- and GDPNP-bound
1 13 Structure and mechanism of formation of the ~ys-~RNA'"-EF-TU-GDPNP ternary cornplex
1.1 -3.1 Structure of E. co/i ~RNA~~' 20
1.1 -4 Techniques generally used to detect binding between
vi i aa-tRNA and EF-Tu-GTP
115 Interaction of EF-Tu with naturally-occurring aminoacylated elongator tRNAs and unacylated tRNA
1.1 -6 EF-Tu-GTP association with tRNA aminoacylated with a non-natural (chemically-modified) amino acid
1.1 -7 Chernical modification of the cysteinyl side chain in CYS-~RNA~~
1-2 Materials
1-3 Experimental Procedures
1.3.1 Partial purification of E. coli cysteinyl-tRNA synthetase from transformed JM1 O1 cells
1.3.2 Preparation of denatured E. coli cysteinyl-tRNA synthetase
1-3.3 Purification of T. thermophihs EF-Tu(NHis6) from transformed JMI 09 cells
1-3.3.1 Method 1 (free EF-Tu)
1-3.3.2 Method 2 (resin-bound EF-Tu)
1.3.4 Purification of E- coli EF-Tu(CHiss) from transformed JMI09 cells
1.3.5 Aminoacylation of ~RNA'" with L-cysteine
1.3.6 Urea-PAGE (with a i0% 8 M urea/TBE poiyacrylarnide gel) with ethidium bromide staining for visualization of tRNA
1.3.7 Autoradiography of 10% 8 M urea/TBE polyacrylarnide gels with bands that correspond to radiolabelled tRNA
1-3.8 Fluorescence imaging of fluorescently-Iabelled CYS-tRNA="
1.3.9 Chemical hydrolysis of the aminoacyl ester bond in cy~tein~l-t~~~~~ 1.3.1 0 Chemical modification of C~S-~RNA~~~with [1 -'4~]-iodoacetamide, IAF, AIASS, and IAEDANS
1.3-11 Ternary complex formation and detection
1-3.11 .l Method 1 - EF-Tu-GDP binding assay
1.3.1 1.2 Method 1 - EF-Tu-GTP binding assay
1-3.1 1.3 Method 2 - EF-Tu-GDP binding assay
1-3.1 1 -4 Method 2 - EF-Tu-GTP binding assay
1-3.1 2 Chemical hydrolysis of EF-Tu-GTP-protected C~S-~RNA'"
1-3.1 3 Molecular modelling
1-3.1 3.1 Structure of the binding site cavity
1.3.1 3.2 Structure of the chemically-modified amino acid
1.4 Results and Discussion
1-4.1 Synthesis of ~~stein~1-t~~~~~
1-4.2 Characterization of ~~steinyl-t~~~~~~
1.4.3 Comparison between DTT and TCEP in terms of their abilities to reduce disuiphide-linked C~S-~RNA~~~
1.4.4 Stability of the aminoacyl ester bond in C~S-~RNA~~~
1-4.5 Stability of the aminoacyl ester bond in EF-Tu-GTP- bound cys-t~NACYI
1-4.6 Characterization of chemicaily-modified CYS-~RNA~~
1-4.7 Interaction of [35~]-~ys-t~~~CFand chemically-modified C~S-~RNA~~~with EF-Tu(NHisô)-GTP and EF-Tu(NHiss)-GDP 1.4.8 The nature of the amino acid binding site in EF-Tu
1-5 References
Chapter 2 Reverse-Phase High Performance Liquid Chromatography of Partially-Purified Chemically-Modified C~S-~RNA~~';
2.1 Introduction
2-2 Materials
2.3 Fxperimental Procedures
2.3.1 Synthesis of IAF-labelled, AIASS-labelled, IAEDANS-labelled, and BIADD-labelled ~~stein~l-t~~~~~
2.3.2 Characterization of BIADD-labelled C~S-~RNA~~~ by Northern blot
2.3.3 RP-HPLC conditions for fractionation of crude chernically-modified C~S-~RNA~~
2-3-3.1 Fractionation of crude IAF-labelled C~S-~RNA~"and characterization of the peaks in the chromatogram
2.3.3.2 Fractionation of crude BIADD-labelled ~ys-~RNA'" and characterization of the peaks in the chromatogram
2.3.3.3 Fractionation of crude AIASS-labelled and IAEDANS-labeiled C~S-~RNA~~~and characterization of the peaks in the chromatograms
2.4 Results and Discussion
2.4.1 Characterization of chemically-modified C~S-~RNA~~
2.4.2 Fractionation of crude BIADD-labelled C~S-~RNA~~ and characterization of the peaks in the chromatogram
2.4.3 Fractionation of crude IAF-labelled C~S-~RNA'~ and characterization of the peaks in the chromatograrn
2-4.4 Fractionation of crude AIASS-labelled and IAEDANS-labelled C~S-~RNA~~~and characterization of the peaks in the chromatograrns
2.5 Summary
2-6 References
Chapter 3 Towards ~RNA'~'-M~~iated Labelling of Firefly Luciferase with Non-Natural Reporter Groups
3.1 Introduction
3.1 -1 Incorporation of fluorescent non-natural amino acids into proteins (applications)
3.1 -2 Techniques for biosynthetic incorporation of non-natural amino acids into proteins (in vitro)
3.1 -2.1 The classic Raney-Nickel experiment
3.1 -2.2 Chemical modification of the a-amino graup in aminoacyl-tRNA
3.1.2.3 Chemical modification of the &-amino graup in lysyl-t~~~Lys
3.1.2.4 Non-sense suppression
3.1 -2.5 Frarne-shift suppression
3.1 -3 Techniques for biosynthetic incorporation of non-naturat arnino acids into proteins (in vivo)
3.1 -3.1 In vivo incorporation by injecting synthetic arninoacyl-tRNAs into Xenopus laevis oocytes 3.1 -3-2 tn vivo incorporation with an orthogonal tRNNaminoacy1-tRNA synthetase pair in E. coli
3.1 -4 Adaptability of non-natural amino acids to the ribosome in E. coli and rabbit reticutocyte
3.1 -4.1 Investigations using puromycin analogues
3.1 -4-2 Library screening by frame-shift suppression
3.1 -5 A novel method for site-specific labellin of proteins with non-natural reporter groups (tRNAgF-media?& protein labelling)
3.2 Materials
3.3 Experimental Procedures
3.3.1 Partial purification of E. coli cysteinyl-tRNA synthetase from programmed JMI O1 cells and preparation of denatured CysRS
3.3.2 Aminoacylation of ~RNA~~'with L-cysteine
3.3.3 Synthesis and purification of BIADD- and IAF- labelled ~~stein~1-t~~~~~
3.3.4 TNT@rabbit reticulocyte lysate coupled transcription/ translation reactions
3.3.4.1 Protein synthesis reaction programmed with plasrnid for luciferase or a CFTR fragment and supplemented with [35~]-methionine
3.3.4.2 Protein synthesis reaction programrned with plasmid for luciferase and supplemented with either [35~]-cysteineor exogenously forrned [35~]-~ys-t~~~CF
3.3.4.3 Protein synthesis reaction programrned with plasmid for luciferase and supplemented with BIADD-labelled C~S-~RNA~", IAF-labelled C~S-~RNA~~~,or ~ranscend~~tRNA 3-3.5 Analysis of translation products 253
3.3.5.1 Autoradiography (using autoradiographic film) 253
3.3.5.2 Autoradiography (using a phosphorimager) 254
3-3.5.3 Fluorescence imaging 254
3-3-54 Western blot 254
3.3.5.5 Photographic luciferase assay 256
3.3.6 Determination of translation protein yield in a rabbit reticulocyte lysate coupled transcription/translation reaction programmed with the piasmid construct for luciferase or a CFTR fragment and supplemented with [35~]-methionine
3-4 Results and Discussion
3.4.1 Protein yields from rabbit reticulocyte lysate coupled transcription/translation reactions programmed with the genes for luciferase and CFTR fragments and supplemented with [35~]-rnethionine
3.4.2 Incorporation of [35~]-c~steineinto luciferase using exogenously formed [3 ~]-cystein~l-t~~~~~
3.4.3 Translations with ~ranscend~~~RNA and BIADD- labelled ~ys-~RNA'"
3.4.4 Translation with IAF-labelled C~S-~RNA~"
3-5 Summary
3.6 Future Directions
3.6.1 Synthesis of chemical ly-modified C~S-~RNA~~'with hydrophobic labels
3.6.2 Factors that could improve incorporation efficiency
3.6.3 Cell-free production of milligram quantities of labelled proteins 3.6.4 t~~~'"-rnediatedmutagenesis for introduction of multiple Ruorophores and for studies on membrane protein structure
3.7 References
Appendix A Buffer Compositions
xiv List of Tables
Table 1-1 Half-lives for the arninoacyl ester bond in ~~stein~1-t~~~~~at different pHs, ternperatures, and buffer compositions.
Table 1.2 Reported half-lives for 19 natural aminoacyl- tRNAs at pH 8.6 and 37 OC.
Table 1.3 Half-lives for EF-Tu-GTP-protected C~S-~RNA~" at different pHs, temperatures, and buffer compositions.
Table 2-1 HPLC solvent and gradient conditions for preparation of RX-C8 column prior to sample injection.
Table 2.2 HPLC solvent and gradient conditions for fractionation of crude IAF-labelled C~S-~RNA~~~.
Table 2.3 HPLC solvent and gradient conditions for fractionation of crude B IADD-labelled C~S-~RNA~?
Table 2.4 HPLC solvent and gradient conditions for fractionation of crude AIASS-labelled and IAEDANS-labelled C~S-~RNA~".
Table 2.5 HPLC solvent and gradient conditions for removal of NaCl from the RX-C8 column,
Table 3.1 Yields of translation products produced in 50 pL rabbit reticulocyte lysate coupled transcription/translation reactions programmed with plasmids pPOLY(A)-luc (SP6), SNCFl2, SNCF56, N2R, N3R, N4R, N5R, NCFII, and pBQ6.2. List of Schemes
Scheme 1.1 (A) Functionalities that are potentially reactive towards a thiol-containing compound. (B) Conversion of a thiol group to an amino group with N-(P-iodoethyl)trifluoroacetamide. (C) Functionalities that are potentially reactive towards an amine-containing compound.
Scheme 1.2 Reaction of cysteinyl-t~~~~~~with N-(1-oxyl- 2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetarnide (A), N-((6-biotinamido)hexyl)-3'-(2'-pyridyldithi0)- propionamide (B), and 1-biotinamido-4-(4'- (rnaleimidomethyl)cyclohexane-carboxamido) butane (C).
Scheme 1.3 Reaction scheme for preparation of C~S-~RNA'"from E. coli ~RNA~~,L-cysteine, ATP, and E. coli CysRS. When the aa-tRNA forms, the amino acid is attached to the 2'-OH of the 3' termina1 adenosine of the tRNA.
Scheme 1.4 Reaction scheme for alkylation of the cysteinyl side-chain of C~S-~RNA~~~by IAF (A) and derivatization of 4-thiouridine by an iodoacetamide functionality (8).
Scheme 2.1 Reaction between NBT and BClP (A), and between napthol AS-MX phosphate and Fast Red TR salt (B), as catalyzed by alkaline phosphatase.
Scheme 3.1 Desulfhydration of cysteinyl-~RNA'~with Raney-Nickel produces alanyl-t~~~~~.
Scheme 3.2 Conversion of ~~steinyl-t~~~~~~to cysteic acid-t~~~'~by treatment with periodate.
Scheme 3.3 Strategy for incorporation of a coumarin- labelled methionine amino acid at the N-terminus of proteins by the use of CPM-SAC-M~~-~RNA~~~.
Scheme 3.4 Site-specific protein labelling by the use of chemically-modified lysyl-t~~~~~'. Scheme 3.5 Outline of non-natural amino acid mutagenesis by amber suppression,
Scheme 3.6. Outline of synthesis of an aminoacyl çuppressor tRNA based on E. coli ~RNA~'~~.
Scheme 3.7 Frame-shift suppression versus in-frame translation in frame-shift suppression mutagenesis. Nucleotide and amino acid sequence of a mutated streptavidin. Underlines indicate stop codons which appear when one of the four-base codons is decoded as a triplet by endogenous E. coli arginyl tRNA.
Scheme 3.8 (A) Strategy for unnatural amino acid incorporation into membrane-bound proteins in intact Xenopus laevis oocytes. The unnatural amino side (L-configuration) side-chain is denoted as R'. (B) Structure of the nonsense suppressor tRNA (derived from yeast ~RNA'~~)designed to maximize suppression efficiency and minimize reacylation by endogenous oocyte aminoacyl-tRNA synthetases.
Scheme 3-9 Outline of a novel non-natural amino acid mutagenesis technique based on the use of chemically-modified C~S-~RNA~~~ (t~~A'"-rnediatednon-natural arnino acid mutagenesis),
Scheme 3.10 Outline of a novel non-natural amino acid mutagenesis technique based on the use of chemically-modified C~S-~RNA"'~ (t~~~"~*-mediatednon-natural amino acid mutagenesis).
Scheme 3.1 1 Potential route for synthesis of chemically- modified ~~stein~l-t~~~~~~constructs with non-polar reporter groups (using the CPM labelling reagent as an example). List of Figures
Figure 1.1 Cartoon representation of the process whereby an aa-tRNA is inserted into the ribosomal A-site during chain e!ongation during protein synthesis in E- coli.
Figure 1-2 Cartoon representation of the relative positions of the A-site tRNA (aa-tRNA) and P-site tRNA (peptidyl-tRNA) in the ribosome during chain elongation during protein synthesis in E. col.
Figure 1 -3 Cartoon representation of the cycling of EF-Tu-GTP during chain elongation during protein synthesis in E. coli.
Figure 1.4 Peptide bond formation between the a-amino group of the aa-tRNA and the acyl group of the peptidyl-tRNA during the peptidyl transferase rsaction during protein synthesis in E. cok
Figure 1.5 Cartoon representation of the translocation of the peptidyl-tRNA and mRNA relative to the ribosome by one codon during chain elongation during protein synthesis in E coli.
Figure 1.6 Cartoon representation of GDP-bound (A) and GTP-bound (B) EF-Tu. Colour codes: yellow = domain 1; green = domains 2 (left) and 3 (right); red = switch I ("effector loop"). Switch II (helix 8) is represented by the yellow helix located between domains 1 and 3, which is rotated by 45" when EF-Tu-GDP is converted to EF-Tu-GTP.
Figure 1.7 (A) Clover leaf representation of the structure of E. coli ~RNA'". Colour codes: orange = 3'-CCA moiety and acceptor stem; red = D arrn and loop; green = anticodon arm and loop; purple = variable loop; yellow = WC (or T) arm and loop; grey = tertiary interactions. Sequence numbering adopted from that for yeast ~RNA'~~.(B) L-shape representation of the structure of E. coli ~RNA'?
xviii Figure 1-8 Structural formulae of modified bases pseudouridine (Y) (A), dihydrouridine (D) (B), 4-thiourid ine (s4u)(C) , and 2-methylthio-N6-isopentenyl-adenosine (rnsiA37) (D).
Figure 1-9 Cartoon representation of the temary complex consisting of T. aquaticus EF-Tu, GDPNP, and C~S-~RNA~~~.The structure was obtained from the RSCB protein data bank (entry code 1B23) and drawn using Prepi.
Figure 1.1O Close-up view of the amino acid-binding cleft in C~S-~RNA~~~-EF-TU-GDPNP.The structure was obtained from the RSCB protein data bank (entry code 1B23) and drawn using Prepi.
Figure 1.1 1 Cartoon showing the hydrogen bond interactions between the aminoacyl ester group of the aminoacyl-tRNA and the adjacent EF-Tu amino acid side chains and main chain amide and carbonyl groups in domain 2 in C~S-~RNA~~~-EF-TU-GDPNP(a) and P~~-~RNA~~~-EF-TU-GDPNP (b). Colour codes: red = domain 1; green = domain 2; orange = 3'-CCA moiety (including amino acid); and grey = EF-Tu amino acid side-chains.
Figure 1-12 Structural formulae of a-aminoisobutyric acid (A) and 1-amino-1 -carboxycyclopentane (B) derivatives of tRNA oligonucleotide analogues.
Figure 1.13 Structural formulae of aminoacyl moieties of phenyllactoyl-t~~~Phe(A) and cinnarnoyl-~RNA'" (8).
Figure 1-14 Photograph of Coomassie brilliant blue R-250-stained gel from an SDS-PAGE (12.5% polyacrylamide) analysis of the partially-purified cysteinyl-tRNA synthetase prep (section 1.3.1). (Lane a = protein molecular weight markers: 175 000,83 000, 62 400,47 500, 32 500, 25 000, 16 500, 6500; and lane b = partially purified CysRS (1 pL aliquot (-2 pg- protein) was loaded). Figure 1.15 Photograph of a Coomassie brilliant blue R-250-stained gel from an SDS-PAGE (12.5% polyacrylamide) analysis of the "pre-induction" control (lane b), "post-induction" control (lane c), "total-lysate" control (lane ci), "fiow-through" sample (lane e), "washn sample (lane f), and selected eluted fractions (lanes g - i) from the purification of T. thermophilus EF-Tu(NHiss) by method 1 (section 1-3.3.1 ). Aliquots of 5 pL were loaded. (Lane a = protein molecular weight markers: 175 000, 83 000, 62 000,47 500, 32 500,25 000, 16 500,6500)-
Figure 1.1 6 Structural forrnulae of the 3' end of L-C~S-~RNA~~ after reaction with 5-iodoacetamidofluorescein (IAF) (a), unreacted 4-acetamido-4'-((iodoacety1)- amino)stilbene-2',2'-disulfonic acid-(AIASS ) (b), unreacted 5-((((2-iodoacetyl)amino)ethyl)- amin0)naphthalene-1-sulfonic acid (IAEDANS) (c), and unreacted (+)-biotinyl-iodoacetamidyl-3,6- dioxaoctanediamine (BIADD) (d). The sulphur atom of the Cys side-chain displaces the iodide atom of the reagent when the adduct forms.
Figure 1.17 A line graph for production of C~S-~RNA'" (using scheme 1.3) using different amounts of cysteinyl-tRNA synthetase (A) and at different incubation tirnes (B).
Figure 1-1 8 (A) Polaroid picture of an ethidiurn bromide-stained 10% 8 M urea/TBE polyacrylamide gel. Lane a: ~RNA'" treated with [35~]-cysteine,ATP, and native CysRS; and lane b: ~RNA'~treated with [35~]-cysteine,ATP, and denatured CysRS. Aliquots of 1 pL were loaded (section 1.3.5). (B) Autoradiograph of (A).
Figure 1.1 9 Line graph for amount of intact [35~]-~ys-t~~~Cys remaining at different time intervals during chernical hydrolysis: (A) at pH 4.5 and 4 OC (e), at pH 8.3 and 4 OC (i);(B) at pH 4.5 and 22 OC (O), at pH 8.3 and 22 OC (=); and (C) at pH 7.3 and 31 OC.
Figure 1.20 Line graph for amount of intact ~5~]-~ys-t~~~CF-~~-~u-~~~temary complexes remaining at different time intervals during chemically hydrolysis ai pH 8.0 and 4 OC (A), 24 OC (B), and 31 OC (C).
Figure 1.21 A composite of an autoradiograph (lanes a and b) and a fluorescence image (lanes c - g) of a 10% 8M urea/TBE polyacrylamide gel. Lane a: [35~]-~ys-t~~~CFbefore treatment with TCEP; lane b: after treatment with TCEP; lane c: (IAF)-labelled C~S-~RNA~~~stained with ethidiurn bromide; lane d: unaminoacylated ~RNA'" stained with ethidium bromide; lane e: (IAF)-treated C~S-~RNA~"(unstained); lane f: (1AEDANS)-treated C~S-~RNA~~~(unstained); and lane g: unarninoacylated ~RNA'" treated with -1AF (unstained).
Figure 1.22 Fluorescence emission scans of labelled tRNAs (solid line) together with unlabelled controls (broken line) (unaminoacylated ~RNA'" treated with the labelling reagents under the same conditions as in the experiments with C~S-~RNA'~~)(approx. 0.5 pM each). (a! IAF- labelled C~S-~RNA~"in a buffer containing 50 mM Tris, pH 7.5 (HCI), 30 mM KCI, and 10 mM MgCI2 (excitation at 492 nm); (b) AIASS-labelled C~S-~RNA~~in a buffer made up of 10 mM NaOCOCH3, pH 4.5 (CH3COOH) (excitation at 329 nm); and (c) IAEDANS-labelled C~S-~RNA~~~in a buffer consisting of 10 mM NaOCOCH3, pH 4.5 (CH3COOH) (excitation at 336 nm).
Figure 1.23 Eiution profiles from GDP-(blue) and GTP-bound (red) EF-Tu binding assays for [1-14~]-acetamido-~ys-t~~~Cys. The concentrations of EF-Tu and tRNA construct in the 1 mL binding reaction were -1 1 PM and -1 -5PM, respectively. Fractions 1 - 21 were collected during coIumn washes while fractions 22 - 31 were collected during eiution.
Figure 1.24 Elution profiles from GDF(blue) and GTP-bound (red) EF-Tu binding assays for IAF-C~S-~RNA~~~.The concentrations of EF-Tu and tRNA construct in the 1 mL binding reaction
xxi were -21 pM and -1 -5PM, respectively. Fractions 1 - 21 were collected during colurnn washes while fractions 22 - 31 were collected during elution.
Figure 1-25 Elution profiles from GDP- (blue) and GTP-bound (red) EF-Tu binding assays for AIASS-rnodified C~S-~RNA~".The concentrations of EF-Tu and tRNA construct in the 1 mL binding reaction were -21 pM and -1 -5pM, respectively. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution.
Figure 1-26 Elution profiles from GDP- (blue) and GTP-bound (red) EF-Tu binding assays for IAEDANS-labelled C~S-~RNA~?The concentrations of EF-Tu and tRNA construct in the 1 mL binding reaction were -21 pM and -1 -5 PM, respectively. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution.
Figure 1.27 Elution profiles frorn E. coli (A) and T. thermophilus (B) GDP- (blue) and GTP-bound (red) EF-TU binding assays for [3ç~]-~ys-t~~~C? The concentrations of EF-Tu and tRNA construct in the 1 rnL binding reaction were -7 1 pM and -2 PM, respectively, for the experiment with E. coli EF-Tu; and 21 pM and -2 PM, respectively, for the experirnent with -T. thermophilus EF-Tu. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution.
Figure 1-28 Elution profiles frorn GTP-bound EF-Tu binding assay for [35~]-~ys-t~~~CyS.The concentrations of EF-Tu and tRNA construct in the i mL binding reaction were -21 pM and -0.2 HM, respectively. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution.
Figure 1.29 Detection of ternary complexes. Fluorescence emission scans (excitation as in figure 1.22) of C~S-~RNA~"derivatives associated with EF-TU-GTP (solid line) (a = IAF-C~S-~RNA~~, b = AIASS-C~S-~RNA~~,and c = IAEDANS-C~S-~RNA~~') eluted from Ni-NTA columns as described in experimental procedures (section 1-3.1 1 -4). EF-TU-GDP control is shown in each case (broken line), measured under identical conditions. Some residual fluorescence from the elution buffer is observed between 450 and 550 nm in al1 cases. A11 scans are averages of three independent binding experiments. Panel d shows counts per minute associated with [35~]-cysteinein the EF-Tu-GTP (solid) and EF-Tu-GDP (open) samples.
Figure 1.30 Structures of the amino acid binding pocket of EF-TU complexed with P~~-~RNA'"(A) and fluorescein-labelled C~S-~RNA'" (B). The protein is shown in surface representation and the terminal adenosine group with the amino acid esterified at the 3' position is shown in stick representation. Atom colours are blue (nitrogen), white (carbon), red (oxygen), and yellow (sulphur). Hydrogens are truncated for clarity. The chemicaI structure of the fluorescein derivative is shown in figure 1-1 6a. The rest of the tRNA molecule extends to the right frorn the terminal adenosine group but is removed for clarity.
Figure 2.1 Structural formulae of the 3' end of lysyl-~RNA~~labelled with p-azidobenzoic acid (ABA) (A), 5-azido-2-nitrobenzoic acid (AN B) (B), 4-(3-trifluoromethyldiazirino)benzoic acid (TDBA) (C), biotin (D), N-biotinyl-6- aminohexanoic acid (AhxBio) (E), and 6-(7- nitrobenz-2-oxa-l,3-diazol-4-yl)arninohexanoic acid (NBDAhx) (F).
Figure 2.2 Structural formulae of the 3' end of CPM-SAc- M~~-~RNA~@'(A), mercapt~acet~l-M~~-~RNA~ (B), disulphide-linked mercaptoacetyl- M~~-~RNA~"(Chand BOD IPY-FL-labelled methionyl-tRNA et (D). figure 2.3 (A) Northern blot of a 10% 8 M urea/TBE polyacrylamide gel showing L~S-~RNA~~'tagged with biotin (~ranscend~~tRNA) (control) (a) and C~S-~RNA~~tagged with biotin (B IADD-labelled ~ys-tRNACF)(b). Unacylated ~RNA- treafed with the biotin Iabelling reagent BIADD was analyzed on the same blot but no band was detected (c). Each band corresponds to -28 pmol of labelled tRNA. (B) ~ranscend~~tRNA structure (3' end only).
Figure 2.4 Typical chromatogram from fractionation of 100 pL of a crude sample containing -28 pM BIADD-labelled C~S-~RNA~~~by RP-HPLC on an RX-C8 column using the solvent and gradient conditions specified in table 2.3.
Figure 2.5 . (A) Polaroid picture of an ethidium bromide- stained 10% 8 M urea/TBE polyacrylamide gel showing nucleic acid composition in the fractions that corresponded to the peaks in the HPLC chromatogram for crude BIADD-labelled C~S-~RNA~~~(figure 2.4). Lane a: biotin-~~s-t~~~~~~(control); lane b: fractions corresponding to the B-PI (8 - 12 min) peak; Iane c: fractions corresponding to the B-P2 peak; lane d: fractions corresponding to the B-P3 peak; and lane e: fractions corresponding to the B-P4 (40 - 45 min) peak. (B) Western blot showing detection of biotin in ~rançcend~~tRNA (lane a) and the B-P2 (lane b) and B-P3 (lane c) fractions. No biotin was detected in the B-PI (8 - 12 min) and B-P4 (40 - 45 min) fractions. The association of biotin with disulphide-linked C~S-~RNA~~~in lanes b and c appears to be due to non-specific binding of al kaline phosphatase-conjugated streptavidin (see section 2.4.2).
Figure 2.6 Typical chromatogram from fractionation of 50 pL of unaminoacylated ~RNA'" (-1 00 PM) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions specified in table 2.3.
Figure 2.7 Typical chromatograrn from fractionation of
xxiv 10 pL of a crude sample containing unlabelled c~s-~RNA'~,unaminoacylated ~RNA~~~,and disul phide-linked C~S-~RNA'~~(-40 pM total tRNA) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions specified in table 2.3-
Figure 2.8 Polaroid picture of an ethidium bromide-stained 10% 8 M ureaîTBE polyacrylamide gel showing nucleic acid composition in the fractions that corresponded to the major peaks in the HPLC chromatograms for unaminoacylated ~RNA'~' (figure 2.6) (lanes a and b) and a crude sample containing unaminoacylated ~RNA'", unlabelled C~S-~RNA'~~,and disulphide-linked C~S-~RNA'~ (figure 2.7)(lanes c and d). Lane a: fractions corresponding to the BU-Pl (6 - 12 min) peak; lane b: fractions corresponding to the BU-P2 (40 - 45 min) peak; lane c: fractions corresponding to the BC-PI (6 - 14 min) peak; and lane d: fractions corresponding to the BC-P2 (40 - 45 min) peak.
Figure 2.9 Typical chromatogram from fractionation of 100 pL of a crude sample that contained IAF-labelled C~S-~RNA~"(-28 PM) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions outlined in table 2.2.
Figure 2.10 A) Polaroid picture of an ethidium bromide- stained 10% 8 M urealTBE polyacrylarnide gel showing nucleic acid content in the fractions corresponding to the peaks in the HPLC chromatogram for crude IAF-labelled C~S-~RNA'"(figure 2-9). Lane a: fractions corresponding to the FI-PI (6 - 14 min) peak; lane b: fractions that corresponded to the FLP2 (15 - 20 min) peak; lane c: fractions corresponding to the FI-P3 (21 - 34 min) peak; and lane d: fractions that corresponded to the FLP4 (40 - 45 min) peak. (B) Fluorescence image of the unçtained gel showing presence of fluorescein in the Fi-PI (6 - 14 min) (lane a), FI-PZ (15 - 20 min) (lane b), FI-P3 (21 - 34 min) (lane c), and FI-P4 (40 - 45 min) (lane d) fractions.
Figure 2.1 1 Typical chrornatogram frorn fractionation of 25 pL of unaminoacylated ~RNA'" (-1 00 PM) by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions outlined in table 2.2,
Figure 2.1 2 Typical chromatogram from fractionation of 10 pL of a crude sarnple containing unlabelled C~S-~RNA~~~,unaminoacylated ~RNA'", and disulphide-linked cys-t~~ACY+(-40 pM total tRNA) by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions specified in table 2-2.
Figure 2.13 Polaroia picture of an ethidium bromide- stained 10% 8 M urea/TBE polyacrylarnide gel showing nucleic acid content in the fractions that corresponded to the major peaks in the chrornatograms for unaminoacylated ~RNA'" (figure 2.1 1) (lane a) and a crude sample containing unaminoacylated ~RNA'~~, unlabelled C~S-~RNA~~,and disulphide-linked ~ys-~RNA'"(figure 2.12) (lane b). Lane a: fractions that corresponded to the FU-P2 (15 - 34 min) peak; and lane b: fractions from the FC-Pl (7 - 14 min) peak.
Figure 2.14 Typical chromatogram from fractionation of 50 pL of a crude sample that contained -28 . pM AIASS-labelled cys-~RNA'" by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions specified in table 2.4.
Figure 2.1 5 Typical chromatogram from fractionation of 10 pL of -1 00 pM unaminoacylated ~RNA'~' by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions specified in table 2-4.
Figure 2.16 Typical chromatogram from fractionation of 10 pL of a crude sarnple with unlabelled cys-~RNA'", unaminoacylated ~RNA'~', and disulphide-lin ked C~S-~RNA~~(-40 PM total tRNA) by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions specified in table 2.4.
Figure 2-17 A) Poiaroid picture of an ethidium bromide- stained 10% 8 M ureafTBE polyacrylamide gel showing nucleic acid content in the fractions corresponding to the peaks in the HPLC chromatogram for crude AiASS-labelled C~S-~RNA~~~(figure 2.14). Lane a: fractions corresponding to the S-Pl (8 - 14 min) peak; and lane b: fractions corresponding to the S-P2 (30 - 35 min) peak. (5) Fluorescence image of the unstained gel showing presence of AIASS-labelled C~S-~RNA'" in the S-Pl (8 - 14 min) fractions. No fluorescence was detected in the S-P2 (30 - 35 min) fractions.
Figure 2.18 Typical chromatogram from fractionation of 5 pL of a crude sample containing IAEDANS-labelled C~S-~RNA~~(-28 PM) by RP-HPLC on an RX-C8 colurnn using the solvent and gradient conditions outlined in table 2.4.
Figure 2.1 9 A) Polaroid picture of an ethidium bromide- stained 10% 8 M urea/rsE polyacrylamide gel showing nucleic acid content in the fractions corresponding to the peaks in the HPLC chromatogram for crude IAEDANS-labelled C~S-~RNA~~~(figure 2.18). Lane a: fractions corresponding to the D-PI (8 - 14 min) peak; and lane b: fractions corresponding to the D-P2 (30 - 35 min) peak. (B) Fluorescence image of the unstained gel showing presence of IAEDANS-labelled C~S-~RNA~~in the D-Pl (8 - 14 min) fractions. No fluorescence was detected In the S-P2 (30 - 35 min) fractions.
Figure 3.1 Structural formulae for the 3' termini of N-acylaminoacyl-tRNAs that have been successfully employed to introduce reporter groups into proteins.
Figure 3.2 Non-natural amino acids (L-configuration) that have been successfully incorporated into proteins by cell-free amber suppression non-natural arnino acid mutagenesis.
Figure 3-3 Non-natural amino acids (L-configuration) that have been successfully incorporated into proteins by cell-free amber suppression NNAAM (continued.. .). The fluorescent residues are nam2d.
Figure 3-4 Fluorescent and other aromatic non-natural amino acids (L-configuration) successfully incorporated into streptavidin by frame-shift suppression mutagenesis.
Figure 3.5 Unnatural amino acids (L-configuration) incorporated into the nicotinic acetylcholine receptor (AchR) binding site (A) and neurokinin-2 (ND) receptor (6)in Xenopus laevis oocytes. (C) Other unnatural amino acids successfully introduced into rnembrane- bound proteins in Xenopus oocytes. The fluorescent residue is fabelled.
Figure 3.6 (A) Structural formulae for unmodified puromycin. (B) Puromycin analogues in which the p-methoxyphenylalanyl group is replaced with S-alkyl- and S-aryt-cysteinyl rnoieties-
Figure 3.7 L-amino acids tested for adaptability to the E. coli ribosome via puromycin analogues. The carboxyl group forms an amide bond with the 3'-amino group of puromycin (Iike that shown in figure 3.68).
Figure 3.8 Model for predicting adaptability of aromatic non-natural amino acids (L-configuration) to the ribosomal A-site, as determined from E. coli cell-free protein synthesis inhibition experiments using 3'-N-aminoacyl analogues of puromycin. Non-natural amino acids (of the type shown in figure 3.7) with benzene rings in regions marked B, E, G, H, and I can adapt, whereas those carrying benzene rings in the other regions are not tolerated.
Figure 3.9 Aromatic non-natural amino acids (L-configuration unless otherwise indicated) tested for detectable incorporation eficiencies in the E. coli S30 extract and rabbit reticulocyte lysate coupled transcriptionltranslation systems.
Figure 3.10 Hypothesis suggested by Hohsaka et al. of allowed and forbidden regions for a benzene ring in L-arylalanine-type amino acids (see figure 3.9 for examples) for acceptance by the E. coli and rabbit reticulocyte ribosomes. Benzene rings in region A: allowed; region B: may be allowed; and C: rejected.
Figure 3.1 1 A) Autoradiographic detection of radiolabelled proteins synthesized in 25 pL rabbit reticulocyte lysate coupled transcriptionltranslation reactions supplemented with [35~]-methionine and programmed with plasmids N2R (lane a), N3R (lane b), N4R (lane c), N5R (lane d), NCFll (lane e), pPOLY(A)-luc (SP6) (lane f), SNCF56 (lane j), and SNCF1 2 (lane k). No radiolabelled protein was detected when the pBQ6.2 plasmid (which encodes full-length CFTR) (lane 1) or no plasmid (lane g) was included in the coupled transcription/translation reaction. From each experiment, a 1 pL aliquot was analyzed. Lanes h and 1 contain protein molecular weight markers. (B) Luciferase activity (lane a), as detected with a photographic assay. No activity was observed when pPOLY(A)-luc (SP6) was not included in the protein synthesis reaction (lane b). Aliquots of 5 pL from the reaction mixtures were assayed.
Figure 3.12 Autoradiographic detection of radiolabelled proteins synthesized in 25 pL rabbit reticulocyte lysate coupled transcriptionltranslation reactions programmed with the plasmid for luciferase and supplemented with 2 pL of [35~]-cysteine. Concentration and specific activity, respectively, for the [35~]-cysteinestocks: 0.01 mM and 2.22 X 1O' dpmlnmol (lane a); 0.1 mM and 2.22 X 1o6 dpmlnmol (lane b); 0.1 mM and 4.22 X 1o6 dpmlnmol (lane c); 0.1 mM and 5.42 X 1o6 dpmlnmol (lane d); 0.1 mM and 7.1 2 X 1o6 dpmlnmol (lane e); 0.1 mM and 8.62 X 1o6 dpmlnmol (lane f); 0.1 mM and
xxix X 1o7 dpmfnmol (lane g). From each experiment, a 15 pL sample was analyzed, No luciferase protein was detected when no plasmid was included in the coupled transcription/translation reaction.
Figure 3.13 Autoradiographic detection of radiolabelled proteins synthesized in a 25 pL rabbit reticulocyte Iysate coupled transcription/ translation reaction programmed with the plasmid for luciferase and supplemented with either 4 pL of free [35~]-cysteine(45 pM at 6.66 X 1o6 dpm/nmol) (lane a) or 4 pL of [3S~]-~ys-t~~~CyS(1 8 pM at 6.66 X 1 o6 dpmfnmol) (lane c). From each experiment, a 5 pL sample was analyzed. No luciferase protein was detected when no plasmid was included in the transcription/trans/ationreaction supplemented with the free hot cysteine (lane b).
Figure 3.14 Autoradiographic detection of radiolabelled proteins (A) and western blot detection of biotinylated proteins (B) synthesized in a 25 PL rabbit reticulocyte Iysate coupled transcriptionltranstation reaction prograrn med with the plasmid for luciferase and supplemented with 2 PL of [35~]-rnethionine (1000 Cilmmol) and 1 pL of ~ranscend~~tRNA (28 pM) (lane a); 2 pL of BIADD-labelled C~S-~RNA~~(species with the 18 min retention time) (14 PM) (lane c); or 0.7 pL of BIADD-labelled C~S-~RNA~~~(species with the 22 min retention time) (41 pM) (lane d). No luciferase protein was detected when no plasrnid was included in the transcription/translation reaction (lane b). From each experiment, a 5 pL aliquot was analyzed. (Biotinylated protein markers: 200 000, 116 000,97 400, 66 200, 45 000,31 000,21 500, and 14 400).
Figure 3.15 Autoradiographic detection of radiolabelled proteins synthesized in a 25 pL rabbit reticulocyte lysate coupled transcription/translation reaction programmed with the plasrnid for luciferase and supplemented with 2 pL of [35~]-methionine(1 000 Cilmmol) and 0.5 pL (lane b); 1 pL (lane c); 1.5 pL (lane d); or 2 pl (lane e) of IAF-labelled C~S-~RNA'~(56 PM). No luciferase protein was detected when no plasmid was included in the transcriptioinf translation reaction (lane a). From each experiment, a 12 pL aliquot was analyzed.
Figure 3.16 Reported fluorescent unnatural amino acids (L-configuration) that have been biosynthetically incorporated into proteins.
Figure 3.1 7 Reported fluorescent unnatural amino acids (L-configuration) that have been biosynthetically incorporated into proteins (continued.. .).
Figure 3.1 8 lodoacetarnide compounds to be tested in t~~~'~'-mediatedNNAAM. List of Appendices
Appendix A Buffer compositions List of AbbreviationslDefinitions
A adenine or adenosine
A260 absorbance at 260 nm
A280 absorbance at 280 nrn a-AC-E-CPM-SAC-LYS- ~RNA~~ L~S-~RNA~"in which the a-NH2 is acetylated and the &-NH2is tagged with CPM-SAC (CPM-labelled- mercaptoacetyl group)
synthetase enzyme that aminoacylates tRNA with an amino acid aa-tRNA aminoacylated tRNA aa-tRNA-EF-Tu(CHis6)-GTP ternary complex consisting of aa-tRNA, EF-Tu(CHis6), and GTP aa-t RNA-EF-Tu-GTP ternary complex consisting of aa-tRNA, EF-Tu, and GTP
ABA p-azidobenzoic acid acetamido-C~S-~RNA~~ iodoacetamide-label led C~S-~RNA'" acetarnido-~~s-t~~~~~- EF-Tu-GTP ternary complex consisting of iodoacetamide- labelled C~S-~RNA~~~,EF-TU, and GTP
AChR acetylcholine receptor
AhxBio N-biotinyl-6-aminohexanoic acid
4-acetarnido-4'-((iodoacetyl)amino)stilbene-2,ZJ- disulphonic acid
AIASS-CYS-~RNA~"- EF-Tu-GTP ternary complex consisting of AIASS-labelled C~S-~RNA~~,EF-TU, and GTP
AK acetyl kinase
alanyl-~RNA*'~ ~RNA*'~aminoacylated with alanine
alany1-t~~~~~~ ~RNA'~aminoacylated with alanine
la-t RNA*'~ ~RNA*'~aminoacylated with alanine
AI~-~RNA~~~ ~RNA~~aminoacylated with alanine
amber UAG stop codon
aminoacyl-tRNA aminoacylated tRNA
aminoacyl-tRNA synthetase synthetase enzyme that aminoacylates tRNA with an amino acid
ANB 5-azido-2-nitrobenzoic acid
AP acetyl phosphate
APS ammonium persulphate
Arg arginine
Asn asparagines
AS~~RNA*'" ~RNA*'" arninoacylated with aspartate
ATP adenosine 5-triphosphate
BD cellulose benzoylated diethylarninoathyl cellulose
BCIP 5-bromo-4-chloro-3-indoiyl phosphate
B IADD (+)-biotinyl-iodoacetamidyl-3,6- dioxaoctanediamine
1-biotinamido-4-(4'- (maleirnidomethylcyclohexane-carboxamido) butane N-((6-biotinamide) hexyl)-3'-(2'pyridyldithio)- propionamide
PME beta-mercaptoethanol
BODIPY-FL 4,4-difiuoro-5,7-dimethyl4bora-3a14a-diaza-s- indacene propionic acid
BOD IPY-FI-methionyl- ~RNA~~' rnethi~n~l-t~~~~"labelled at the a-amino group with BODIPY-FL
cystic fibrosis
CFTR cystic fibrosis transmembrane conductance regulator
[l-14~]-iodoacetamide radiolabelled iodoacetamide
CK creatine kinase
CP creatine phosphate
cpm count per minute
CPM 7-diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin
CPM-SAC-M~~-~RNA~~'
CysRS synthetase enzyme that aminoacylates ~RNA~~' with cysteine
cystein yl-~RNA'" ~RNA'" aminoacylated with cysteine
cysteinyl-tRNA synthetase synthetase enzyme that aminoacylates ~RNA'" with cysteine
~RNA'~~aminoacylated with cysteine
~ys-~RNA'"-EF-TU-GDPNP ternary complex consisting of C~S-~RNA~~~~EF-TU, and GDPNP
xxxv ternary complex consisting of C~S-~RNA'~,EF-Tu, and GTP
C~S-~RNA"~~ ~RNA"'* aminoacylated with cysteine
D dihydrouracil or dihydrouridine
DDTG dithiodiglycolic acid
DEAE diethylaminoethyl
DMSO dimethylsulfphoxide
dpm disintegration per minute
DTT dithiothreitoi
E rnolar extinction coefficient
E-ABA-L~S-~RNA~F ABA-labelled lysyl-t~~~LYS
E-A~XB~O-L~S-~RNA~~~ AhxBio-labelled L~S-~RNA~~~
&-AN B-L~S-~RNA~" ANB-modified L~S-~RNA~~*
E-B~O-L~S-~RNA~~ biotin-labelled L~S-~RNA"
E. COIÏ Escherichia colÏ
EDTA ethylenediaminetetraacetate
EF-TS elongation factor - temperature stable
EF-TU eiongation factor - temperature unstable
EF-Tu(CHis6) EF-Tu fused with C-terminal hexa-histidine tag
EF-TU-GD P GDP-bound EF-Tu
EF-TU-GDPNP GDPNP-bound EF-Tu
EF-TU-GTP GTP-bound EF-Tu
EF-Tu(NHis6) EF-TU fused with N-terminal hexa-histidine tag EF-Tu(N Hiss)-GTP
E-N B DA~X-L~S-~RNA~~~
E-TD BA-L~S-~RNA~F fMet f~et-tRNA'M~~ ~RNA~~'aminoacylated with N-forrnylmethionine
FRET fluorescence resonance energy transfer
G guanine or gusmsioe
G-domain GTP/GDP-binding domain
GDP guanosine 5'-diphosphate
GDPNP nonhydrolyzable analogue of GTP
GlnRS glutarninyl-tRNA synthetase
Glu glutamate glutarninyl-tRNA synthetase synthetase enzyme that aminoacylates ~RNA~'" with glutamine
GIU-t RNA~'" ~RNA~'"aminoacylated with glutamate
G IU-t RNA='" ~RNA~'"aminoacylated with glutamate
G~Y glycine g~y~y~-t~~~G~Y~RNA~'~ aminoacylated with glycine
G I~-~RNA~IY ~RNA~'~aminoacylated with glycine
G-protein GTP/GDP-binding protein
GTP guanosine S'-triphosphate h hour
H-bond hydrogen bond
HEPES acid
His histidine
His6 hexa-histidine tag
His-tag hexa-h istidine tag
HPLC high performance liquid chromatography
1- iodide
IAEDANS 5-((((2-iodoacetyl)amino)ethyl)amino)naphthaIene 1-suIphonic acid
ternary complex consisting of IAEDANS-labelled C~S-~RNA~~~,EF-TU, and GTP
IAF
ternary complex consisting of IAF-labelled C~S-~RNA~~,EF-TU, and GTP
IE-TFA N-(P-iodoethyl)trifluoroacetamide
IF-2 initiation factor named IF-2
IIe-t RNA"~ ~RNA"~aminoacylated with isoleucine lPTG isopropyl P-D-thiogalactopyranoside
Kd equilibrium dissociation constant ko bs pseudo first order rate constant
LB medium Luria-Bertani medium
L~u-~RNA~~" ~RNA~~'arninoacylated with leucine
LSC liquid scintillation counting LYS lysine
LysRS synthetase enzyme that aminoacylates ~RNA~~' with lysine
L~s-~RNA~~~ ~RNA~~aminoacylated with lysine
I~S~I-~RNA~~ ~RNA~~aminoacylated with lysine lysyl-tRNA synthetase synthetase enzyme that aminoacylates ~RNA~~' with lysine
M~~-~RNA~"tagged at the N-terminus with a mercaptoacetyl group
Met methionine rnethi0n~1-t~~~~~~~RNA~~~ aminoacylated with methionine methionyl-~RNA~~' tRhIAMe'aminoacylated with methionine rnethionyl-tRNA synthetase synthetase enzyme that aminoacylates ~RNA~~' (or ~RNA~~')with methionine
synthetase enzyme that arninoacylates ~RNA~~' (or ~RNA~~')with methionine
M~~-~RNA~~' ~RNA~"aminoacylated with methionine
M~~-~RNA~~' ~RNA~~'aminoacylated with methionine min minute
M. jannachii Methanococcus jannachii mRNA messenger RNA msiA
MW molecular weight
MWCO molecuiar weight cut-off
~-acet~l-val~l-t~~~~~'~al~1-t~~~~~' tagged with an N-acetyl group N-acylaminoacyl-tRNA aminoacyl-tRNA in which the a-amino group is acylated
methionyl-~RNA~~'in which the a-amino group is acylated
NBDAhx 6-(7-nitrobenz-2-oxa-I ,3-diazol-4- y1)aminohexanoic acid
3-N-(7-nitrobenz-2-oxa-1 ,3-diazol-4-YI)-2,3- diaminopropionic acid
NBT nitroblue tetrazolium
~-form~lmethionyl-t~~~~~~rnethi0n~1-t~~~~~~ tagged with an N-forrnyl group
Ni-NTA nickel-nitrilotriacetic acid
NK2 neurokinin-2 receptor
NNAAM non-natural amino acid mutagenesis ochre UAA stop codon
0 Dsoo optical density at 600 nrn
2'-O H 2'-hydroxyl group
3'-O H 3'-hydroxyl group
OIN overnight opal UGA stop codon
O-tRNA orthogonal suppressor tRNA pCysS2 E. co/i cysteinyl-tRNA synthetase plasmid
PEG
PEP phosphoenolpyruvate peptidyl-tRNA tRNA connected to a peptide via the 3'-OH of the terminal adenosine L-p-fluoro-~he-tRN~'~~ L-P~~-~RNA'~=tagged with a pfluoro group
Phe phenylalanine
phenylalanyl-t~~~phe ~RNA'" arninoacyiated with phenylalanine
P~~-~RNA'~= ~RNA'~~aminoacylated with phenylalanine
P~~-~RNA~"-EF-T~-GDPNPternary complex consisting of P~~-~RNA'", EF-TU, and GDPNP
phenylalanyl-tRNA Synthetase synthetase enzyme that aminoacylates ~RNA'~~ with phenylalanine
Pi inorganic phosphate
PK pyruvate kinase
PK~ negative log of the acidity constant K,
plasmid for EF-TU fused with C-terminal hexa-histidine tag
P~O-~RNA~" ~RNA'" aminoacylated with proline
PVDF polyvinylidene difluoride
QY quantum yield
RNase ribonuclease
RP-HPLC reverse phase HPLC
rPm revolutions per minute
RRL rabbit reticulocyte lysate rt room temperature
RT retention time s second rnercaptoacetyl-~et-t~~~~
S-cerevisia e Sacharomyees cerevisiae
[35~]-~ys/cysteine radiolabelled cysieine
[35~]-~ys-t~~~cpl ~~steinyl-t~~~~~radiolabelled cysteinyl-~RNA'"
SDS-PAGE sodium dodecyl sulphate - polyacrylamide gel Electrophoresis
s~c-~RNA'~' ~RNA'~'aminoacylated with selenocysteine
~elenoc~st~l-t~~~~~~~RNA'~'aminoacylated with selenocysteine
Ser serine
s~~-~RNA'~~ ~RNA'~'aminoacylated with serine
[35~]-~etlmethionine radiolabelled methionine
s4u 4-thioudridine
T 5-methyluridine
t1/2 half-1ife
T- aquaticus Thermus aquaticus
TBE Tris-borateIEDTA
TBS Tris-buffered saline
TBSS TBS with 7% SDS
TBSST TES with 0.5% ~ween@20
TCA trichloroacetic acid
TCEP tris(2-carboxyethy1)phosphine
TDBA 4-(3-trifluoromethyldiazirino)benzoicacid
TEMED N, N, N',NY-tetramethylethylenediamine
xiii TM transmernbrane
TMR tetramethylrhodarnine
TNS 6-(p-to1uidinyl)naphthalene-2-sulfonic acid
~ranscend~~tRNA biotin-labelled lysyl-t~~~LYS
Tris tris(hydroxyrnethyl)aminomethane tRNA transfer RNA
~RNA*'~ alanine-specific tRNA
~RNA~~~ arginine-specific tRNA
~RNA~"" asparagine-specific tRNA
~RNA~F cysteine-specific tRNA
~RNA~~~ eubacterial initiator (methionine-specific) tRNA t RNA='" glutamine-specific tRNA
~RNA~I"~ an isoacceptor of glutamine-specific tRNA t RNA~I~ glutamate-specific tRNA t RNA~'~~ an isoacceptor of glycine-specific tRNA t RNAII~ isoleucine-specific tRNA
~RNA~~' leuci~e-specifictRNA
~RNA~~ lysine-specific tRNA
~RNA~@' elongator methionine-specific tRNA
~RNA~~~ phenylalanine-specific tRNA
~RNA~~O proline-specific tRNA
~RNA~~~ seienocysteine-specific tRNA t RNA'" serine-specific tRNA tyrosine-specific tRNA
cysteine-specific tRNA with a UCA (anti-opal) anticodon t RNA""' valine-specific tRNA
T~P tryptophan
T. thermo philia Tetrahymena thermophila
7. thermophilus Thermus thermophikis tyrosyl-tRNA synthetase çynthetase enzyme that aminoacylates ~RNA~~~ with tyrosine tyr~~y~-t~~~Ty ~RNA~~'aminoacylated with tyrosine
TyrRS tryosyl-tRNA synthetase
T~~-~RNA~~ ~RNA~~'arninoacylated with tyrosine
U uracil or uridine urea-PAGE urea - polyacrylamide gel electrophoresis val-~RNA'~' ~RNA'~'aminoacylated with valine valyl-t RNA'~~ ~RNA'~~aminoacylated with valine va1~1-t~~~~~' ~RNA'~'aminoacylated with valine w-C Watson-Crick
Y pseudouridine Chapter 1
Interaction of Fluorescently-Labelled C~S-~RNA~~*with EF-Tu-GTP 1.i Introduction
1.i .1 Significance of EF-Tu in biosynthetic incorporation of fluorescent non-natural amino acids into proteins by non- sense suppression
The introduction of fluorescent groups at specific sites in proteins is a powerful strategy for obtaining information on the cellular localization of, structure of, and dynamics of proteins under near-native conditions (1). With the developrnent of techniques for detecting single fluorescent molecules in biological systems (2-4), the impetus for developing general methods for biosynthetic incorporation of fluorophores is heightened. One promising general method for site-specific labelling of proteins with fluorescent groups is the biosynthetic incorporation of fluorescent non-natural amino acids via non-sense suppression (5-7). In general, the incorporation of chemically-modified arnino acids into proteins at a predetermined position can be accomplished by adding zppropriately constructed suppressor tRNAs to an E. coii (~scherichiacoli) cell- free protein-synthesizing system that has been programrned with a gene bearing a stop codon at the site of interest (8-1 1). The amino acid is introduced by suppression of the premature termination codon by the aminoacylated suppressor tRNA. To understand the strength and limitation of this approach for site-specific labelling, a brief review of the process of protein biosynthesis with emphasis on the role of EF-TU (elongation factor - temperature unstable) is required, 1.1.1.1 General overview of protein synthesis in E. coli with emphasis on the role of EF-Tu
Biosynthesis of proteins, which is often referred to as translation, occurs in three distinct stages: translation initiation, chain elongation, and translation termination (12).
Protein synthesis initiation involves formation of a translation complex
(12). In an E. coli cell, the complex consists of the messenger RNA (mRNA) template to be translated, ribosomal subunits 50s and 30S,initiator aminoacyl- tRNA (aa-tRNA) ~-form~lmethion~l-t~~~~~~(f~et-t~~~~~'),and several accessory proteins cailed initiation factors. The assernbly ensures that the correct initiator codon (AUG), which occurs at a specific location at the beginning of the mRNA, and thus the correct reading frame (that which gives rise to the desired protein) are selected before translation begins. Because of the AUG codon, al1 biosynthesized proteins begin with a methionine (Met) residue- Post- translational processing occurs to remove the methionine where necessary.
During chain elongation, amino acids are joined to a growing polypeptide chain in a pre-determined order that corresponds to the order of codons in the mRNA template) (12). The amino acids are appended via a three-step microcycle. The steps in the microcycle are: 1) positioning of the correct aa- tRNA in the A-site of the ribosome (as specified by an mRNA codon); 2) formation of a peptide bond behveen the new amino acid and the nascent polypeptide chain (a peptidyl transferase reaction); and 3) shifting of the mRNA and peptidyl-tRNA relative to the ribosome by one codon (translocation).
Concomitant with the translocation process is dissociation of the deaminoacylated tRNA from the ribosome. The growing polypeptide chain is synthesized from the N-terminus to the C-terminus,
Insertion of the proper arninoacyl-tRNA into the ribosomal A-site is catalyzed by a temperature-unstable elongation factor caIled EF-Tu (elongation factor - temperature unstable) (figure 1.1 ) (22-16). In the cyctoplasml EF-Tu forms a ternary complex with one molecule of guanosine 5'-triphosphate (GTP) and the aa-tRNA (aa-tRNA-EF-Tu-GTP). The ternary complex diffuses to the ribosome and makes contacts with the A-site. If a mismatch occurs between the anticodon of the aa-tRNA and the codon of the mRNA, the ternary complex is rejected and thus leaves the ribosome and re-enters the cyctoplasm. However, if correct base-pairing is detected between the codon and anticodon, the complex is repositioned such that favourable contacts are made by EF-TU-GTP (GTP- bound EF-Tu) with the aminoacyl-tRNA (also known as the A-site tRNA), the tRNA carrying the growing polypeptide chain (also referred to as the peptidyl- tRNA or P-site tRNA), and the ribosomal subunits in the vicinity) (12). These contacts, as a whoie, trigger hydrolysis of GTP to GDP (guanosine 5'- diphosphate) and Pi (inorganic phosphate). EF-Tu-GDP (GDP-bound EF-TU) does not form favourable contacts with the ribosome or tRNAs and as a result leaves the ribosome and enters the cytoplasrn) (12). At the same time, the aa- tRNA molecule is released from the elongation factor and inserted completely into the A-site, next to the P-site tRNA (figure 1.2) (12). The two tRNA constructs are now ready for the peptidyl transferase reaction. A utc
Figure 1.1. Cartoon representation of the process whereby an aa-tRNA is inserted into the ribosomal A-site during chain elongation during protein synthesis in E. coli (Picture obtained from Moran et al. text (12)).
In the cytoplasm, inactive GDP-bound EF-Tu is converted back to an
active GTP-bound state with assistance from a second elongation factor, which is
temperature-stable, called EF-Ts (elongation factor - ternperature stable) (figure
1.3) (72). After a GTP-bound EF-Tu is regenerated, the elongation factor binds
another aminoacyl-tRNA moiecule. A GTPIGDP cycling step is mandatory as
EF-Tu-GDP is unable to bind arninoacylated tRNA.
During the peptidyl transferase reaction, the a-amino group of the
aminoacyl-tRNA in the ribosomal A-site forms a peptide bond with the acyl group
of the peptidyi-tRNA in the ribosomal P-site (figure 1-4) (12). The carbonyl
carbon of the acyl group undergoes a nucleophilic attack by the amino group and
the P-site tRNA is displaced. The new polypeptide chain, which has increased
by one amino acid, becomes attached to the A-site tRNA.
After peptide bond formation, the deaminoacylated P-site tRNA exits the
ribosome via the E (exit) site, while the mRNA template and the new peptidyl-
tRNA shift relative to the ribosome by one codon. In this manner, the next codon
of the mRNA is exposed in the ribosomal A-site for translation. This process is
called translocation and is catalyzed by a third elongation factor called EF-G
(figure 1.5) (12).
The three-step microcycle is repeated every time a new codon is translated.
Translation is terminated when the translation cornplex reaches the end of the coding region in the mRNA (12).. Termination is prompted when the codon in the ribosomal A-site is a termination triplet of UAG (amber), UGA (opal), or UAA
H-c-R.,, 1 NH t O=C I H-C-R. I HS '$
P silc r RNA $ O f o=p-~Q
I CH:
H O Ott I c=o I H-C-R,.: I HN 1 c=o 1 H-C-R..I l NH I
Figure 1.4. Peptide bond formation between the a-amino group of the aa-tRNA and the acyl group of the peptidyl-tRNA during the peptidyl transferase reaction during protein synthesis in E-coli. (Picture obtained from Moran et al. text (12)). Figure 1.5. Cartoon representation of the translocation of the peptidyl-tRNA and mRNA relative to the ribosome by one codon during chain elongation during protein synthesis in E. coli (Diagram obtained from Moran et al. book (12)). (ochre). ln this situation, a release factor, rather than an aminoacylated elongator tRNA, recognizes the codon and triggers a cascade of reactions, which ultimately causes release of a newly synthesized peptide and dissociation of the translation cornplex,
With the amber suppression technique, a variety of non-standard amino acids with novel chernical, structural, and spectroscopy (e-g. fluorescent) properties have been introduced into proteins (8, 17, 18). For certain residues which were rejected or weakly tolerated by the bacterial translational machinery
(e-g.homoglutamate, cyclopropylglycine, and omithine), poor recognition of non- natural aa-tRNAs by the GTP-bound form of elongation factor Tu has been cited as a possible reason for the failure of the non-natural amino acid mutagenesis
(NNAAM) procedure (5, 19-22). If a non-natural aminoacyl tRNA is prepared and then introduced to a translation system, the first step that must occur is the binding of the aa-tRNA to EF-Tu-GTP.
1.1.1 EF-Tu background
1.1.2.1 Function of EF-Tu
Elongation factor Tu's primary role is binding and delivery of aminoacylated elongator tRNA to the ribosome during chain elongation during protein synthesis (12). Consistent with this function is the fact that almost al1 cellular aa-tRNA molecules are found in ternary complexes with the protein and
GTP (23). In addition to this role, however, the elongation factor carries out nurnerous other processes during chain elongation. Elongation factor Tu selectively binds aminoacylated elongator tRNAs with
L-aminoacyl groups, while discriminating against uncharged tRNAs (13, 24, 25), naturally occurring mischarged tRNAs (e.g. AS~-~RNA*'"(in Thermus
Thermophilus (T. fhermophilus)) (26) and lu-~RNA~'" (in Gram-positive bacteria,
Archaebacteria, mitochondria, and chloroplast) (27)), aa-tRNAs with a D- configuration (28, 29), and aminoacyl-tRNAs that are substrates for other translation factors, such as initiator fl'vlet-t~~~~~'(30) and selenocystyl-~RNA'~'
(S~C-~RNA'~~)(31 , 32). The discrimination against the mischarged tRNAs, fMet-
~RNA~~~,and S~C-~RNA'~~ prevents misincorporation of the arnino acids into proteins. Recognition of aa-tRNA by EF-Tu-GTP involves the amino acid moiety and the acceptor stem (see section 1.1 -3)(33, 34). The lack of binding when
~RNA*'" or ~RNA~'"is mischarged with Asp or Glu, respectivey, has been suggested to result from an unfavourable presentation of the arnino acid by the noncognate tRNA because of the negatively charged side chain carboxyl group of Asp or Gln and structural peculiarities of the acceptor arm (26, 27). By virtue of its unique nucleotide sequence and structure, f~et-tff~~~~'is recognized by a
GTP-bound initiation factor calied IF-2 (initiation factor - 2) and therefore participates in the assembly of the translation initiation complex (12, 30). The aminoacylated elongator tRNA rneth~on~1-t~~~~~~(M~~-~RNA~='),which has a different nucleotide sequence and overall structure, but the same anticodon as its initiator counterpart, is recognized by EF-TU-GTP. The antideterminant of Sec-
~RNA'~'for binding on EF-TU involves solely the length of the acceptor stem (35, 36); this aminoacyl-tRNA associates specifically with a special elongation factor called SelB (31, 37). IF-2 and SelB are homologous to EF-Tu (31, 38, 39).
When an aminoacylated elongator tRNA is bound in a temary complex, elongation factor Tu is found to protect the aminoacyl ester bond that links the arnino acid to the tRNA from spontaneous chemical hydrolysis, which could otherwise occur under physiological conditions (15, 34, 40)-
It has also been suggested that GTP-bound elongation factor Tu is responsible for catalyzing the transacylation of the aminoacyl group in a bound aminoacyl-tRNA from the terminal adenosine 2'-hydroxyl (2'-OH) to the 3'- hydroxyl (3'-OH) group (41). The 2'-to-3' transacylation step is mandatory because the 3' isomer is required for peptidyl transferase (12) during chain elongation during protein synthesis.
In addition to its conventional role in protein synthesis, elongation factor
Tu has been found to catalyze several other types of reactions in vitro which are not directly related to chain elongation. For example, Blumenthal and co-workers discovered in 1972 that the protein is one of the subunits of the QP replicative complex, an enzyme responsible for replication of the RNA from the E. coli bacteriophage QP (42). In 1970, Travers and colleagues found that the elongation factor also serves as a transcription factor, which preferentially stimulates synthesis of ribosomal RNA (43). Discoveries in 1976 by Jacobson and collaborators (44) and in 1991 by Young and Bemlohr (45) that elongation factor Tu is also associated with the inner surface of the plasma membrane and becomes methylated when an E. coli cell culture is deprived of an essential nutrient (e-g. ammonia) suggested the possibility of a specific role in regulation of growth, possibly as a principal component of an unidentified signal transduction pathway in bacteria. Substantiating this view is the fact that EF-Tu is a G-protein
(GTP-binding protein). GTP hydrolysis is usually linked to a regulatory function
(46) and G-proteins are generally recognized as playing the role of "on" and "off" switches in a myriad of signalling processes in any organism (47, 48). In 1998,
Richarme et al. reported that the elongation factor displays a protein-disulphide isomerase activity (49). Protein-disulphide isomerase activity comprises disulphide bond formation, disulphide bond reduction, and disulphide interchange or isomerization. Which function occurs depends on the imposed redox potential and the nature of the polypeptide substrate (49). However, the biological significance of a protein disulphide isomerase activity from EF-Tu has not been evaluated. Lastly, evidence has been reported by Kudlicki and colleagues in
1997 (50) and Caldas and collaborators in 1998 (51 ) that show elongation factor
Tu could act as a rnolecular chaperone. In particular, it was observed that EF-Tu could increase the rate of refolding of unfolded proteins, protect proteins against thermal denaturation, and form complexes with unfolded proteins and not with folded proteins.
1.1.2.2 Structure of GDP- and GDPNP-bound EF-TU
EF-Tu is a monorneric protein with a rnolecular weight (MW) of 43 000 and
406 amino acid residues. It is present at a very high level in E. coli cells and is the most abundant protein in bacteria. With approximately 135 000 molecules per cell, which corresponds to a cyctoplasmic concentration of 100 - 200 PM, the elongation factor comprises 5 -1 O% of total cell protein, a level which is equivalent to that of aa-tRNA and in vast molar excess over other essential protein components of the translation machinery (12, 52-54). A huge abundance is required presumably to allow elongation factor Tu to cany out al1 its tasks during protein synthesis.
The high dernand for EF-Tu is accommodated by the existence of two separate EF-Tu genes in the bacterial chromosome (52, 55). The genes are termed tufA and tufB. The products of the two genes differ by a single amino acid and are virtualty indistinguishable in terms of function (56).OnIy the C- termini of the products are different, with the tufA product having a glycine (Gly) and the tufs product containing a serine (Ser).
EF-Tu is N-terminally acetylated, while a non-conserved lysine (Lys) residue at position 56 can be monomethylated or dimethylated (38, 57).
EF-Tu was the first protein to be identified as a GTP!GDP-binding protein.
It is structurally similar to the G-proteins found in the membranes of eukaryotic cells, suggesting that they al1 evolved from a common ancestral protein. In fact, it has been suggested that EF-Tu is probably the unique ancestor of al1 G- proteins given its central and highly conservative role in translation (34). The elongation factor was the first G-protein to be structurally investigated.
A break-through in EF-Tu structurai characterization occurred in 1985 when la Cour and colleagues (581, along with Jurnak and CO-workers(59), elucidated the first structural details of the GDP-binding domain of E. coli EF-Tu- GDP. This set the stage for a number of reports on the structure of the
eiongation factor in several different functional stages in the following years.
In 1993, Berchtold and colleagues determined a structure for the active
EF-Tu-GDPNP from T. thermophilus (60),while Kjeldgaard et al. provided structural details for the active EF-Tu-GDPNP from Thermus aquaticus (T. aquaticus) (61 ). GDPNP is a non-hydrolyzable analogue of GTP and EF-TU associates with the two cofactors with virtually the same afFinity (62).
Polekhina and collaborators in 1996 reported the X-ray structure for T. aquaficus GDP-bound EF-Tu (63),while the high resolution structure for EF-Tu-
GDP from E. co/i. was reported in 1996 by Abel and CO-workers(64) and in 1999 by Song and colIeagues (65).
EF-Tu-EF-Ts dimers from E. coli and T. thermophilus were structurally elucidated in 1996 by Kawashima et al. (66) and in 1997 by Wang et al. (67). respective1y.
Models of EF-Tu-GDP and EF-Tu-GTP from Bacillus stearothermophilus
(B. stearothermophilus) based on the X-ray structures just described were also constructed in 1998 by Krasny et al. (68)-
From the X-ray data, much is known about the structure of GDP-bound and GTP-bound EF-Tu.
EF-Tu consists of three domains: domain 1, domain 2, and domain 3
(figures 1.6A and 8) (69). Domain 1 is the GDPfGTP-binding (or GTPase) domain. It consists of 200 amino acids. With a central P-sheet surrounded by a- helices, its secondary structure is typical of a nucleotide-binding domain and is found to be similar to other G-domains (70). Domains 2 and 3 each contain 100 amino acid residues. Both are comprised of P-barrels. They are held together as one structural unit, which is often referred to as domain 2 + 3, by strong interdomain interactions.
There are dramatic differences between the GTP- and GDP-bound forms of the protein. Whereas EF-TU-GDP adopts an open-conformation (figure 1.6A),
EF-TU-GDPNP (which can be considered to be the same as EF-Tu-GTP) takes on a closed-configuration (figure 1.6B). Local conformational changes in two motifs located in domain 1 are primarily responsible for the difference in conformations between GDP-bound and GTP-bound EF-Tu. These two motifs are named switch 1 (figures 1-6A and 6)and switch Il (figures 1.GA and B).
Switch 1, also referred to as the "effector loop", comprises arnino acid residues 51 through 64 (amino acid sequence numbers adopted from that for T. aquaficus
EF-Tu), while switch II contains amino acid residues 83 through 100. When
GDP-bound EF-Tu is converted to its GTP-bound counterpart, the "effector loop" changes from a hairpin structure to a short a-helix (cf. figures 1.6A and B). In the switch II region, an a-helix, termed helix B, shifts along the sequence by approximately four residues, thereby rotating the axis of the helix by about 45'
(cf. figures 1.6A and B). This helix forms part of the interface between domains 1 and 3 (figures 1.6A and B) and thereby explains the change in domain-domain interaction in EF-Tu upon its activation. Furthermore, a change in helix B is directly coupled to the introduction of the y-phosphate group in the GTP-binding site, for this terminal phosphate group induces an almost 180" peptide flip at a conserved glycine just pnor to helix B (not shown). Overall, a domain rearrangement that corresponds to a 90' rotation between domain l and domain
2 + 3 takeç place upon nucleotide exchange (cf. figures 1.6A and B). The more open conformation of the GDP-bound form appears to be more flexible and sensitive to denaturants (e.g. temperature) than the GTP-bound form (69).
1.1.3 Structure and mechanisrn of formation of the C~S-~RNA~~~-EF-TU-GDPNPternary cornplex
In 1995, the first crystallographic structure of a ternary complex was solved by Nissen and collaborators (33). The ternary complex consisted of T. aquaticus EF-TU, GDPNP, and yeast phenylalanyl-tRNA (phe-t~~~'")(Phe-
~RNA~~~-EF-TU-GDPNP).Following this work, reports on the solution structures for the same ternary complex and a second complex, which contains E. col; Phe-
~RNA'", rather than the aa-tRNA from yeast, were published in 1998 by Bilgin and collaborators (71). The NMR experiments gave rise to ternary complex structures which were similar to that seen from the crystal. Finally, Nissen and colleagues in 1999 reported the high resolution structure for the ternary complex that consists of T. aquaticus EF-TU, GDPNP, and ~.coli~~stein~l-t~~~~~~ (Cys-
~RNA'~)(C~S-~RNA~Y~-EF-TU-GDPNP ) (34).
The X-ray crystallographic structures for the two ternary complexes Phe-
~RNA'"-EF-TU-GDPNP and C~S-~RNA~~~-EF-TU-GDNPprovided insight into the structural basis for recognition of aminoacylated elongator tRNA by EF-TU-GTP.
Compared with the structure of the complex with P~~-~RNA~",the structure of the complex with C~S-~RNA~~revealed more specific features of a factor-bound tRNA molecule and is of an ail-bacterial temary cornplex- For this reason, the structure of C~~-~RNA~~-EF-TU-GDPNPis elaborated and deviations from that of
P~~-~RNA~~~-EF-TU-GDPNPare mentioned. However, before the details of the structure are discussed, a background description of the structure of E. coli
~RNA'" is necessary.
A .1.3.1 Structure of E. coli ~RNA~~'
~RNA'~'from Escherichia coli consists of 74 nucleotides and has a molecular weight of 24 000 (34, 72). The nucleotides are nurnbered from the 5' end to the 3' end with respect to the 76 nucleotides of yeast ~RNA'" ((figure 1.7A)
(34, 73, 74). This convention was established because yeast ~RNA'~~was the first tRNA for which a high resolution three-dimensional structure was solved.
The secondary structure of E. coli ~RNA'~'consists of a number of stems and loops which are organized into 5 main parts (figure 1-7A). The parts are called the acceptor stem (residues 1 - 9 and 65 - 76) which includes the 3' CCA end, the dihydrouridine (D) arm (residues 1O - 25) which includes the adjacent loop, the anticodon arm (residues 26 - 44) which includes the adjacent loop, the variable loop (residues 45 - 48), and the T (5-methyluridine) (TYC) arm (residues
49 - 64) which includes the adjacent loop (figure 1-7A). With respect to the sequence of yeast ~RNA'", position 17 of the D loop and position 47 of the variable loop are missing for E. coli ~RNA'" (figure 1 -7A). The cysteine-specific tRNA contains a number of modified bases (figure 1-7A). There are three pseudouridine (Y) bases (Y32, Y39, and Y55) (figure 1.8A), two dihydrouridine bases (D20 and D21) (figure 1.8B),one 4-thiouridine (s4u)base (s4u8) (figure
1.8C). and one 2-methylthio-N6-isopentenyl-adenosine (msiA) base (msiA37)
(figure 1-8D) (72, 75). ~RNA~~'has a GCA anticodon, which occupies positions
34, 35, and 36 (figure 1-7A).
In translation, ~RNA'~is capable of interacting with two different codons in an mRNA template, UGU and UGC, both of which code for a cysteine amino acid. Bases C and A frorn the anticodon form standard Watson-Crick (W-C) base-pairs with bases U and G from each of the two codons. Base G from the anticodon is located at a conformationally flexible region in the tRNA, which is known as the wobble position, and thus is able to form hydrogen bonds (H-bond) with two different bases (76). While it forms a wobble base-pair with base U from the UGU codon, it interacts with base C from the UGC codon via conventional W-
C base-base interaction (76).
Three-dimensional structures have not been reported for either unacylated or aminoacylated E. coli ~RNA'". However, the X-ray crystallographic structure for the cysteine-specific tRNA in a ternary complex with EF-Tu and GDPNP (34) provides an almost-complete description of the structure of E. coli ~RNA'~'in a naturally modified state.
Overall, E. coli ~RNA~~adopts the canonical L-shape (figure 1.78 (picture obtained from Stryer Biochemistry text book (77)) (78). The two ends of the L- shape are separated by approximately 73 A (78). The three-dimensional structure of the tRNA is held together by a set of tertiary base-base interactions from bases in the D arm, anticodon arm, and WCarm (figure 1.7A) (34, 78). E coli ~RNA'" contains features within its primary, secondary, and tertiary
structures which are essential for specific recognition by its cognate aminoacyl-
tRNA synthetase. Such elements are called identity elements or identity
determinants. The identity determinants for E. coli ~RNA'~are: 1) the GCA
anticodon (79, 80); 2) the U73 base (which is often referred to as the
discfiminator base) (figure 1.7A) (79-82); 3) the tertiary base-base interaction
between G15 of the D loop and G48 of the variable loop (often designated as the
G15:G48 base pair or "Levitt base pair") (figure 1-7A) (83) (78) (84) (72, 85-87)
(located at the corner of the tRNA L-shape (figure 1-7B)); and 4) the Al3:A22
secondary interaction in the D stem (which interacts with a third base, Ag, to form
an adenosine-rich base triple, A9:A13:A22 (figure 1.7A) (which in turn is
structurally coupled to the G15:G48 tertiary base pair, an interaction that is
important for the orientation of the "Levitt base pair")) (34, 84-87).
For the formation of a ternary cornplex of CYS-~RNA'~,GDPNP, and
elongation factor Tu, al[ three dornains of EF-Tu participate in the binding with
GDPNP and cy~tein~l-t~~~~~(figure 1.9) (34). The structures of the GDPNP-
bound protein and aminoacylated tRNA are not much changed upon formation of the ternary complex. The protein binds to only one end of the aminoacylated tRNA, leaving the rest of the nucleic acid free to interact with the ribosome-
Regions of C~S-~RNA~~that are in contact with the elongation factor in the ternary complex include: 1) the aminoacytated CCA end; 2) the acceptor stem; and 3) the TYC stem plus loop (figure 1.7A) (34). The terminal adenosine and aminoacyi ester bond are accommodated in specific pockets in the P-barre1 of Figure 1.9. Cartoon representation of the ternary complex consisting of T. aquaficus EF-Tu, GDPNP, and ~ys-tRNACyS. The structure was obtained from the RSCB protein data bank (entry code 1B23) and drawn using Prepi. domain 2 (figure 1.10). The side chain of the cysteinyl group is located in a deft
fomed between domains 1 and 2 (figure 1.1 0). The phosphorylated 5' end of the
tRNA is bound in a positively charged surface depression at the intersection
between al1 three dornains (figure 1.9). The two midine nucleotides of the 3'-
terminal CCA moiety and nucleotide residues 1-3 and 73 of the acceptor stem
(figure 1.7A) are in contact with switch I and switch II in domain 1 (figures 1.6 and
1.9). One side of the backbone fold of the WCstem (nucleotide residues 50-54
and 63-67) (figure 1 -7A) forms a large interface with the surface of the P-barre1 of
domain 3 (figure i-9). The anticodon helix, formed by the D (dihydrouridine) arm
and the anticodon arm (figure i.7A), extends from the complex (figure 1.9).
When the overall architecture of C~S-~RNA~"-EF-GDPNPis compared to
that for P~~-~RNA~~~-EF-TUGDPNP,the hostructures are virtually identical.
However, in an EF-Tu-based alignment, the tRNAs do not superimpose exactly.
While regions in direct contact with elongation factor Tu are perfectly matched,
anticodon helices are slightly offset with the ~RNA'"~elbow angle (90') being IO0 tighter than that for ~RNA'~'(1 00') (34).
The aminoacyl ester bond of C~S-~RNA~"is stabilized in domain 2 by a set of hydrogen bonds formed with adjacent amino acid side chains and protein backbone amide groups (figure i .? la) (34).
The 2'-OH group vicinal to the aminoacyl ester bond forms a specific hydrogen bond with a side chain carboxylate from glutamate 271 (Glu271). At the same time, the hydroxyl group accepts an H-bond from a main chain NH from arginine 274 (Arg274). The side chain of this argin ine bends over and forrns an
Figure 1.1 1. Cartoon showing the hydrogen bond interactions between the arninoacyl ester group of the aminoacyl-tRNA and the adjacent EF-TU amino acid side chains and main chain amide and carbonyl groups in domain 2 in Cys-tRNACyS-EF-Tu-GDPNP(a) and Phe-tRNAPhe-EF-Tu-GDPNP(b). Colour codes: red = domain 1; green = domain 2; orange = 3'-CCA (including amino acid) moiety; and grey = EF-Tu amino acid side-chains. (Pictures obtained from Nissen et al. paper (34)). H-bond from its protonated amino group to the carbonyl group of the cysteinyl rnoiety. This is different from what is found in the P~~-~RNA'~~-EF-TU-GDPNP ternary complex, where Arg274 donates a hydrogen bond from its a-NH to the carbonyl group of the phenylalanyl moiety and has a side chain that adopts a fully-extended conformation (figure 1-11 b) (34). It has been suggested that the difference in interaction is probably due to differences in the aminoacyl side groups, which in turn create variations in conformations of the aminoacyl bonas.
Another possibility is that the bulkiness and hydrophobicity of the phenylalanyl side chain may disfavour close contact between the phenylalanyl ester bond and arginyl side chain.
The a-amino group of the cysteinyl moiety simultaneously accepts a hydrogen bond from the main chain amide of histidine 273 (His273)and donates an H-bond to the main chain carbonyl group from asparagine 285 (Asn285). A third hydrogen bond from the amino group to the main chain carbonyl group of
Glu271 was observed in the phenylalanine complex, though it cannot form in the
C~S-~RNA~~~-EF-TU-GDPNPcomplex as the distance between the donor and acceptor is more than 4 A (34). Hydrogen bond formation between the nitrogen atom of the a-amino group of Cys and the main chain amide of His273 prevents protonation of the amino group (which would occur in free solution). The neutral state of the NH2 group gives rise to a decrease in electrophilicity for the carbonyl carbon of the cysteinyl ester bond and thus may decrease the rate of spontaneous chernical hydrolysis that leads to deacylation of C~S-~RNA'"(88- 90). The aminoacy! ester bond is also sterically protected from hydrolysis by the
docking of the aminoacyl moiety into the active site of the elongation factor.
In the cleft formed between domains 1 and 2, the cysteinyl side chain is in
van der Waals contact with Asn285 and His67 (figure 1.l O). In the Phe-tRNA Phe-
EF-TU-GDPNP structure, the asparagine side chain is positioned at a different
location to optirnize packing of the pheny!alanyl side chain (cf. figures l-7 l a and
b)-
The interface between the WCstem helix of C~S-~RNA~~and domain 3
of GDPNP-bound EF-Tu consists of an array of both polar and hydrophobic
interactions of tRNA residues 50-54 and 6347 (figure 1.7A) with adjacent arnino
acid side chains,
Interestingly, it appears that weak interactions between tRNA and dornain
3 of GTP-bound elongation factor Tu prevent selenocystyl-~RNA~~'from forming
a ternary complex with EF-Tu-GTP (34, 37). The structural data suggests that
initiator f~et-t~~~~~'is not a substrate for EF-Tu-GTP for two reasons. First, its
acceptor stem has unpaired bases, which perturbs the association with dornain 1
of the elongation factor. The second reason is that N-formylation of the a-amino
group blocks formation of a hydrogen bond between the nitrogen of the NH2
group and the main chain amide of His273 (12, 34).
1.1.4 Techniques generally used to detect binding between aa-tRNA and EF-Tu-GTP
Several different techniques have been developed to rnonitor interaction between aminoacyiated tRNA and GTP-bound elongation factor Tu in vitro. Virtually al1 methods are based on a physical separation of ternary complexes from free EF-Tu-GTP andfor unassociated aa-tRNA. lsolated ternary complexes are subsequentIy detected usually by Iiquid scintillation counting (LSC) of radiolabelled aminoacylated tRNA or fluorescence spectroscopy of fluorescently- labelled aminoacyl-tRNA. These methods have been used to study ternary complex formation both quaIitatively and by measuring equilibrium dissociation constants (Kd).
One traditionally used method for puriving aa-tRNA-EF-Tu-GTP complexes makes use of hydrophobic nitrocellulose filters which are capable of binding protein, but not tRNA (91-93). When a solution containing ternary complexes is applied to such a filter, EF-Tu-GTP-associated aminoacyl-tRNA passes through and into the filtrate, whereas uncomplexed elongation factor is trapped. Ternary complexes are detected and quantified as the difference in filter-bound radiolabelled EF-Tu in the absence and presence of aa-tRNA.
Gel filtration on a sephadex G-100 colurnn separates aa-tRNA-EF-Tu-
GTP from aa-tRNA-free elongation factor based on molecular size (94).
Separated ternary complexes are characterized by a distinct elution (or retention) time. The aminoacyl rnoieties of the aminoacylated tRNA molecules are radiolabelled so that ternary complex formation can be detected and quantified by LSC.
A third binding assay takes advantage of the fact that factor-bound aa- tRNA constructs are protected from spontanteous chemical hydrolysis (95-97).
Like in the gel filtration assay, the aminoacyl group in aa-tRNA is radiolabelled. In a solution buffered at an alkaline pH, aminoacyl-tRNA molecules that are not bound to EF-TU-GTP are hydrolyzed while those that are protected in ternary complexes remain intact. Ternary complexes of aa-tRNA-EF-Tu-GTP and deacylated tRNA constructs are then separated from free amino acids (from deacylated aa-tRNAs) by filtration on a ~hatman@filter disk. The disk binds the macromolecules but not the free amino acids. Ternary complexes are detected and quantified by liquid scintillation counting of the disks.
Similar to what has just been described is a ribonuclease (RNase)- resistance assay (25, 94, 98-100). The rationale for this assay is based on protection by GTP-bound elongation factor of the 3'-terminal phosphodiester bond (pCpA) of aminoacylated tRNA from enzymatic cleavage by pancreatic ribonuclease. Again, the aminoacyl group of the aa-tRNA is radiolabelled.
Ternary complexes are distinguished from digested tRNA constructs, detected, and quantified by filtration on a ~hatman"filter disk to remove free amino acids followed by liquid scintillation counting.
In a fifth approach, aminoacylated tRNA molecules are tagged with a fluorescent reporter group by chemical modification of a 4-thiouridine or 2- thiocytidine residue (101 -1 04). Association of fluorescently-labelled aa-tRNA constructs with GTP-bound elongation factor Tu is detected and quantified by an increase in fluorescence intensity. Fluorescence intensity increase is suggested to be brought about by conformational changes in aa-tRNA in the vicinity of the fluorescent moiety, induced by binding to the protein. This fluorescence-based assay is the only technique that enables aa-tRNA-EF-Tu-GTP ternary complex detection and Kd measurements in a sample that is actually at equilibrium throughout an experiment-
The most recently developed technique for separating aa-tRNA-EF-Tu-
GTP ternary complexes frorn uncomplexed elongation factor involves construction of a recombinant EF-Tu protein that has a C-terminal hexa-histidine
(Hiss) tag (His-tag) (EF-Tu(CHis6)) (1 05). A his-tag exhibits high affÏnity for nickel ions. When a mixture consisting of aa-tRNA-EF-Tu(CHis6)-GTP ternary complexes, uncomplexed aminoacyl-tRNA constructs, and uncornplexed elongation factor moiecules is applied to a nickel-conjugated column, al1 species containing an EF-Tu(CHis6) rnoiety are trapped by the nickel-affinity column, whereas free aa-tRNAs, which are not associated with a His-tag, are unaffected by the metal and therefore flow through the column- Ternary complexes are eluted from the nickel-affinity matrk by imidazoie. In this experiment, the aminoacylated tRNA is conventionally radiolabelled so that purified ternary complexes are detected and quantified by liquid scintillation counting. Formation of an EF-Tu-GTP affinity-matrix has also been achieved by immobilization of the active elongation factor on cyanogen bromide-activated sepharose (106, 107).
1.1.5 Interaction of EF-TU with naturally-occurring aminoacylated elongator tRNAs and unacylated tRNA
GTP-bound elongation factor Tu Sinds to al1 naturally-occurring aminoacylated elongator tRNAs with nanomolar affinity (25). Equilibriurn dissociation constants for the different aa-tRNA-EF-Tu-GTP ternary complexes
Vary 14-fold and lie in the 1O-'' to IO-' M range (25). The chernical nature of the aminoacyl side-chain was found to affect the strength of interaction of the aminoacyl-tRNA with EF-Tu-GTP (15, 96-98). Differences in affinities of the aminoacylated elongator tRNAs for the elongation factor do not lead to a preferential suppiy of strongly binding aa-tRNAs to the ribosome since the intracellular level of the elongation factor Tu is sufficient to complex al1 aminoacylated tRNAs present in a cell (95). For this reason, the observed differences in temary cornplex formation are most likely to be of no consequence for normal protein synthesis.
Only a weak interaction is observed in vitro between GTP-bound elongation factor Tu and unarninoacylated tRNA (13, 24, 104, 108-1 1O). A Kd >
1o4 M. which corresponds to an association that is 10~-foldweaker than that seen for aminoacylated tRNA, has been reported (105).
Affinity of EF-TU in a GDP-bound state for aminoacylated tRNA is also weak relative to the affinity of the GTP-bound form (24, 104, 109). A & of approximately 1O-' M has been measured (104, 109). This is sufficiently high with the given in vivo concentrations of EF-TU and aa-tRNA (-1 00 PM) (23, 11 1) to allow complex formation between GDP-bound EF-Tu and aa-tRNA, however, in the cytosol. This will, nevertheless, not happen under normal growth conditions since the high concentration of GTP (1 mM) relative to GDP (0.1 mM)
(1 i 2) in combination with weaker dissociation of EF-Tu-GTP relative to the stability of EF-Tu-GDP (1 13) will prevent accumulation of EF-Tu-GDP. The weak zssociation of the GDP-bound form of the elongation factor with aa-tRNA makes possible release of the aa-tRNA from the aa-tRNA-EF-Tu-GDP temary wrnpIex in the ribosomal A-site during chain etongtion during protein synthesis.
EF-Tu-GDP has virtually no detectable affinity for uncharged tRNA (109).
To summarize, for a strong interaction to occur, elongation factor Tu must be complexed with GTP and tRNA must be aminoacylated. When either one requirement is relaxed, there is still an appreciable interaction between protein and nucleic acid. Only when both requirements for strong interaction are absent can an interaction not be detected.
1.1.6 EF-TU-GTP association with tRNA aminoacylated with a non-natural (chemically-modified) amino acid
Elongation factor Tu's primary role in protein synthesis, to deliver the twenty different amino acids aminoacylated to elongator tRNAs to the ribosome, dictates that it has a broad substrate specificity. While EF-Tu has been shown to discriminate against a number of naturally-occurring mischarged tRNAs (e-g.
ASP-~RNA~'"(in T. thermophilus) (26) and GIU-~RNA~'"(in Gram-positive bacteria, Archaebacteria, mitochondria, and chloroplast) (27)) (discussed briefly in section 1.1 -2.1) and aa-tRNAs that are substrates for other translational factors
(30-32) (see section 1.1.2.1), very few direct investigationsof this interaction with tRNAs aminoacylated with non-natural amino acids have been reported (15).
Yamane and CO-workers,in 1981, examined interaction of D-tyrosyl-
~RNA~~'(D-T~~-~RNA~~) with GTP-bound elongation factor Tu (29). They discovered that this aminoacylated tRNA binds with an affinity that is 25-fold lower than that for the corresponding L- enantiomer. EF-Tu has been found to functionally interact with achiral molecules. In
1980 and 1982, Bhuta and colIaborators (114, 115) showed that a-
aminoisobutyric acid (figure 1-1 2A) and 1-amino-l -carboxycyclopentane (figure
1.1 26) derivatives of tRNA oligonucleotide analogues appeared to bind to GTP-
bound elongation factor Tu.
Fahnestock and colleagues, in 1972 (1le), and Derwenskus et al., in
1983) (1 17), discovered that replacement of an aa-tRNA aminoacyl group with
either a phenyllactoyl (figure 1-1 3A) or cinnarnoyl (figure 1.1 35) group, an
analogue that is missing an cc-amino group, leads to a lowering of the efficiency
of EF-Tu-GTP binding by a factor of 300.
Lastly, Pingoud and Urbanke, in 1980, reported that L-p-fluoro-Phe-
~RNA'~~associates with EF-TU-GTP with an affinity that is sirnilar to that for
unmodified ~-phen~lalanyl-t~N~~~~(96).
1.1.7 Chernical modification of the cysteinyl side chah in CYS-~RNA~Y+
A very convenient method of constructing non-natural aminoacylated
elongator tRNAs is to chemically modify the cysteinyl side-chain in cysteinyl-
~RNA'" with different thiol-reactive reagents. The thiol group of cysteine can be
modified with chernicals that contain haloacetamide (iodoacetamide,
bromoacetamide, and chloroacetamide), alkyl halide, maleimide, aziridine,
symmetric disulphide, or thiolsulphonate functionalities (scheme 1.1A) (118).
Reaction with an haloacetamide, maleimide, alkyl halide, or aziridine results in formation of a thioether bond, whereas modification by a disulphide or HO-
Figure 1.12. Structural formulae of cc-aminoisobutyric acid (A) and l-am in04 -carboxycyclopentane (B) derivat ives of t RNA oligonucleotide analogues.
Afkyl hallde or Thloether Haloacetamlde (X = 1, Br, CI)
Maleimide Thloether
Adridine Thioether f) RICON + R~NH* ----) R'C-NH~ + HO !bO Carboxamlde O RIS-SR' + R~SH R'S-SR~ Succlnlmidyl ester Syrnmetric disutphide Mked disulphlde R'SO~CI + R'NH~ - R'SO~-NHR~ t HCI Sulfonyl chloride Sulfonamlde
Thlolsulphonate Mixed dlsulphlde ? + R'NH? R'CH=NR' H20 Reduction R(CH~-NH@ R'CH + ,-w - Schiff base Alkylamlne Alde hyde
Scheme 1.1. (A) Functionalities that are potentially reactive towards a thiol-containing compound. (8) Conversion of a thiol group to an amino group with N-(p-iodoethyl)trifluoroacetamide. (C) Functionalities that are potentially reactive towards an amine-containing compound. thiolsulphonate functionality generates a new mixed dilsulphide cornpound. The
reactions are carried out in a solution with a pH typically between 6.5 and 8.0, so
denaturation and spontaneous degradation of the target rnolecule is not a major
concern. Many thiol-specific labelling reagents are commercially available (e-g.
Molecular Probes Inc. (118) and Toronto Research Chemicals).
The thiol functionality in cys-t~~ACrjcould also be derivatized to contain
a free amino group. Such a conversion could be achieved with the reagent N-(P-
iodoethyl)trifluoroacetamide (IE-TFA) in a one-step process (scheme 1.1 6)(1 19).
This reaction is called aminoethylation. It is highly selective for thiols, so side
reaction with the a-amino group of C~S-~RNA~~shou Id not occur. This reagent
is also commercially available (Pierce). Reporter groups may then be introduced
to the aminoethylated C~S-~RNA~~~by reaction with amino-reactive reagents.
Reactive moieties which are selective for primary amines include isothiocyanate,
succinimidyl ester, sulfonyl chloride, and aldehyde (scheme 1.1 C) (1 18).
Despite the reactivity of the cysteinyl side chain, very few chemically-
modified cys-4~~~~"analogues have been reported- Chapeville and colleagues converted ~~steinyl-t~~~~~~to alanyl-t~~~CYS(A~~-~RNA'~') by reductive desulfhydration with Raney Nickel in 1962 (120). In 1970, Kabat and
CO-workersderivatized C~S-~RNA'" with spin-labelling reagent N-(1-oxyl-2,2,5,5- tetrarnethyl-3-pyrrolidiny1)iodoacetamide (121 ) (scherne 1-2A). Lastly, Ohtsuka and CO-workersin 1997 constructed two different (but similar) biotinylated Cys-
~RNA'" analogues by reaction with thiol-reactive derivatives of biotin (N-((6- biotinamide)hexyl)-3'-(2'-pyridyldithi0)-propionamide ( biotin-HPDP) and 1- Scheme 1.2. Reaction of L-cysteinyl-tRNACYSwith N-(1-0xy1-2,2,5,5-tetramethyl- 3-pyrrolidinyl)iodoacetamide (A), N-((6-biotinam ido)hexyl)-3'-(2'-pyridy1dithio)- propionamide (B), and 1-biotinam ido-4-(4'-(maleim idornethyl)cycIohexane- carboxamido) butane (C). biotinamido-4-(4~-(maleimidomethyl)cyclohexane-carboxamÎdo)butane (biotin-
BMCC)) (schemes 1.2B and Cl respectively) (122). However, none of these non- natural aminoacylated tRNA constructs have been tested for interaction with elongation factor Tu.
The recently reported X-ray structure of C~S-~RNA'~-EF-TU-GDPN P prompted us to explore further the range of Ruorescent, non-natural amino acids that might be tolerated by elongation factor Tu in an effort to determine what, if any, the inherent limitations for the biosynthetic incorporation of fluorophores might be. We examined the binding of a series of L-~ys-~RNA'" constructs carrying fluorescent groups to T. thermophilus EF-Tu-GTP using a Ni-NTA
(nickel-nitrilotriacetic acid) agarose affinity chromatography assay. In the present study, the criteria for choosing a set of fluorescent tags were: photostability, commercial availability, structural diversity, thiol-selectivity, and water solubility.
None of the chemical modifications were found to significantly impair the interaction of the aminoacylated tRNA with EF-Tu. These results can be rationalized by examining the three-dimensional structure of the binding pocket in the ternary complex. 1.2 Materials
E. col; ~RNA'" was purchased from Subriden RNA (Rollingbay, WA). A plasmid encoding E. coli cysteinyl-tRNA synthetase (CysRS) (pCysS2) - constructed by Eriani and CO-workers(123) - was obtained in JMI O1 cells as a gift from Dr. H. Jakubowski (New Jersey Medical School). A plasmid encoding T. thermophilus EF-Tu tagged with an N-terminal hexa-histidine sequence (EF-
Tu(NHis6)) was a gift from Dr. M. Sprinzl (Universitat Bayreuth) (105). A plasmid encoding E. cofi EF-Tu tagged with a C-terminal hexa-histidine sequence (EF-
Tu(CHis6)) (pKECAHis) (124) was a gift from Dr. B. Kraal (Leiden University).
[35~]-~ysteineand [l-14~]-iodoacetamide were purchased from Amersham Life
Science (Baie dJUrfe,Quebec). Fluorescent iodoacetamide derivatives were purchased from Molecular Probes (Eugene, OR). Ni-NTA agarose was purchased from Qiagen (Missisauga, ON). Fluorimetric grade imidazole was purchased from TCI America (Portland, OR). Competent JMI 09 cells were purchased from Promega (Madison, WI). Prestained broad range protein molecular weight markers were purchased from New England Biolabs
(Mississauga, ON). Protein assay reagent was purchased from BIO-RAD
(Mississauga, ON). All other chernicals were purchased from Sigma-Aldrich
Canada (Oakville, ON), BioShop Canada Inc. (Burlington, ON), or the Materials
Distribution Centre (MDC) at the University of Toronto (Toronto, ON). 1.3 Experimental Procedures
1.3.1 Partial purification of E, coli cysteinyl-tRNA synthetase from transformed JMlOl cells
E coli cysteinyl-tRNA synthetase was partially purified from JMl O1 cells
transformed with the pCysS2 plasmid following a slightly-modified version of a
published protocol by Jakubowski (125). Glassware and LB (Luria-Bertani)
medium for cell cultures were autoclaved in an 8000-DSE autoclave (Napco)
according to the manufacturer's instruction before use. Cells were taken from a
single isolated colony on a freshly streaked plate made from agar, LB medium,
and 100 pg/rnL ampicillin (126). Two 10 mL cultures containing JMl O1 cells
transformed with pCysS2, LB medium (126), and 100 pg/mL ampicillin in 50 mL
polypropylene conical tubes were grown for 6 hours (h) at 37 OC in a Series 25
incubator shaker (New Brunswick Scientific Co., Inc., Edison, New Jersey). Each
10 mL culture was then transferred to 300 mL of LB medium containing 100 .
pg/mL ampicillin, in a 1 L Erlenmeyer flask. This second culture was allowed to
grow overnight (O/N) at 37 OC in the incubator shaker. The next morning,
cultures were transferred to 50 mL polypropylene copolymer centrifuge bottles.
The bottles were centrifuged at 4 OC and 10 000 rprn for 10 minutes (min) in a
Sorvall Superspeed RC2-B refrigerated centrifuge. After centrifugation,
supernatants were removed by suction from an aspirator. In a 50 ml
polypropylene conical tube sitting on ice, the two pellets were combined
(approximately 3.2 g in total) and resuspended in 10 mL of a buffer containing 1O mM potassium phosphate, pH 6.8, 1 mM P-mercaptoethanol (PME), snd 10% glycerol (buffer A). The ceil suspension was subsequently sonicated on ice using a VirSonic 60 sonicator (VIRTIS). Sonication was perfomed at half-maximal power (setting 10) such that a 1 min pulse was applied at 30 second (s) intervals for a total of 4 cycles. The sonicated suspension was transferred to a 50 mL centrifuge bottle, which was centrifuged at 10 000 rpm at 4 OC for 1O min. AH procedures after centrifugation were carried out on ice so as to prevent proteolytic degradation and denaturation. After centrifugation, supernatant
(lysate) was recovered and transferred to a 100 mL beaker. Three millilitres of freshly prepared 6% streptomycin sulphate solution was added to the lysate to precipitate nucleic acid. After treatment with streptomycin sulphate, the lysate was transferred to a 50 mlcentrifuge bottle, which was centrifuged at 10 000 rpm and 4 OC for 10 min to pellet precipitated nucleic acid. After centrifugation, the supernatant was transferred to a 100 mL beaker. Protein was then precipitated by ammonium sulphate at 75% saturation. Ammonium sulphate solid (4.76 g) was slowly added to the lysate with constant stirring with a stirring rod to avoid local supersaturation. Ammonium sulphate-treated lysate was transferred to a 50 mL centrifuge bottle so that precipitated protein could be peiletted by centrifugation at 10 000 rpm and 4 OC for 10 min. After centrifugation, the supernatant was discarded by aspiration. The protein was then dissolved in 3 mlof buffer A. The protein solution was divided into 200 pL aliquots. The aliquots were stored in a -20 OCfreezer. SDS-PAGE (sodium dodecyl sulphate - polyacrylamide gel electrophoresis) on a 12.5% polyacrylamide gel was performed to characterize and determine the purity of the protein (figure 1.14). It appeared tom the gel that the synthetase was -80%
pure. Further purification did not increase the functional efficiency of the enzyme
significantly (data not shown). The protein was quantified using a BIO-RAD
protein assay (127). Each 200 PL aliquot was found to have a concentration of 2
mg/mL. When CysRS was required for an experiment, an aliquot was thawed on
ice (-5 min). Immediately afier the amount of protein required was removed, the
stock was transferred back to the freezer.
1.3.2 Preparation of denatured E. coli cysteinyl-tRNA synthetase
To obtain stocks of denatured E. coli cysteinyl-tRNA synthetase for use as
controls in aminoacylation experiments, three 200 PL aliquots of the CysRS
preparation (section ? -3.1) were thawed, transferred to screw-capped
microcentrifuge tubes, and incubated in an 80 OC water bath for 20 min. The
denatured protein was stored in a -20 OC freezer until required for experiments.
When the denatured synthetase was required, a stock was thawed at room
temperature (rt) and then placed on ice. lmmediately after use, the aliquot was
transferred back to the freezer.
1.3.3 Purification of 1. thermophilus EF-Tu(NHis6) from transformed JM109 cells
1.3.3.1 Method 1 (free EF-Tu)
T. thermophilus EF-TU with an N-terminal hexa-histidine tag was purified frorn JM109 cells transformed with the corresponding plasmid following a CysRS (MW = 52 000)
Figure 1.14. P hotograph of Coomassie brilliant blue R-250- stained gel from SDS-PAGE (12.5% polyacrylamide) analysis of the partially-purified cysteinyl-tRNA synthetase prep (section 13.1 ). (Lane a = protein molecular weight markers: 175 000, 83 000, 62 000, 47 500, 32 500: 25 000, 16 500, 6 500; and lane b = partially purified CysRS (1 pL aliquot (-2 pg protein) was loaded). modified version of a protocol reported by Ribeiro and colleagues (105).
Glassware and LB media for ceIl cultures were autoclaved before use. Cells were obtained from a single isolated colony on a freshly-streaked plate containing agar, LB medium, and 100 pg/mL ampicillin. A 20 mL culture containing LB medium, 100 pg/mL ampicillin, and JM109 cells transformed with the plasmid coding for EF-Tu(NHis6) was incubated overnight in a 100 mL
Erlenmeyer flask in a 37 OC incubator shaker. The next morning, 1L of LB medium, supplemented with 100 pg/rnL ampicillin, in a 4 L Erlenmeyer flask, was inoculated with the 20 mL overnight culture. The large culture was allowed to grow in the 37 OC incubator shaker until an optical density at 600 nm (OD~OO)of
0.8 was reached (-5 h). At this point, a 1 mL aliquot was removed and stored in a 4 OC refrigerator for SDS-PAGE analysis at the end of the experiment. This sample was a "pre-induction" control. The rest of the culture was induced with 1 mM freshiy-prepared isopropyl p-D-thiogalactopyranoside (IPTG) and allowed to grow at 37 OC in the incubator shaker for an additional 4.5 h. After 4.5 h, another
1 rnL aliquot was removed and stored in the -20 OC fridge for SDS-PAGE analysis- This aliquot was a "post-induction" control. The culture was transferred to 4 separate 250 mL polypropyIene copolymer centrifuge bottles and cells were harvested by centrifugation at 4 OC and 7000 rpm for 20 min in a Sorvall
Superspeed RC2-B refrigerated centrifuge. The supernatants were discarded by decantation, and the cell pellets, with the bottles, were placed at -20 OC overnight. The next morning, cells were thawed on ice (-1 h). The thawed cells were then combined and suspended in 20 mL of a buffer containing 50 mM NaH2P04, pH 8.0,300 mM NaCI, and 10 mM irnidazole (buffer B). Freshly prepared lysozyme was added to the ceIl suspension at a fina! concentration of 1 mg1mL. Lysozyme treatment was canied out on ice for 30 min, after which, the cell suspension was transferred to two 50 mL polypropylene conical tubes. On ice, the ce11 suspensions were sonicated. Sonication was carried out at half- maximal power with four 1 min pulses at 30 s intervals- The sonicated suspensions were transferred to 50 mL centrifuge bottles and centrifuged at 4 OC and 10 000 rpm for 30 min. The supematants were combined and transferred to a fresh 50 mL polypropylene conical tube and kept on ice while the pellets were discarded. At this point. a 5 pL aliquot of the lysate was removed and stored at -
20 OC for SDS-PAGE analysis. This was a "total-lysate" control. The supernatant was dialyzed using spectra/~or@"molecularporous" regenerated cellulose dialysis membrane with a molecular weight cut-off (MWCO) of 12 000 -
14 000 (2.5 cm width), in a 2 L beaker, againçt 1 L of a buffer containing 50 mM
Tris-CI (tris(hydroxymethyl)aminomethane), pH 7.5, 50 mM NH&I, 50 mM KCI,
10 mM MgCI2, 10 mM PME, and 50 PM GDP (buffer C), with a stirring bar, overnight, and in a 4 OC sliding-door refrigerator. The next morning, the dialyzed lysate was transferred to a 50 mL polypropylene conical tube and combined with
5 mL of Ni-NTA agarose solution (in ethanol). After capping and sealing with parafilm (to prevent leakage), the tube was placed on a rotatory shaker (Roto Mix
Type 51600 Thermolyn (Sybron)) in the 4 OC sliding-door refrigerator for 2 h.
Two hours later, the mixture of lysate and resin was loaded into an empty, capped 10 mL disposable polypropylene column with a frit (Pierce, Rockford, IL). The column was allowed to sit at 4 OC for 45 min undisturbed to allow the Ni-NTA agarose to completely settle at the bottom of the column. After this step, the volume of liquid above the top of the resin was allowed to pass through the column. A 5 pL aliquot of this fiow-through ("flow-throughn sample) was stored at
4 OC for SDS-PAGE analysis. The column was subsequently washed with buffer
B to remove non-specifically bound protein and nucleic acid. Protein and nucleic acid were monitored by UV absorbance at 280 nm (A280)and 260 nm (&O), respectively, using a UWNIS Lambda 2 spectrometer (Perkin Elmer).
Approximately 300 mL of buffer was required to wash al1 the non-specifically bound material from the column and bring the A260and to background values (OD = O - 0.02). A 5 pL aliquot was removed from the wash ("wash" sample) and stored at -20 OC for SDS-PAGE analysis. EF-Tu protein was eluted from the nickel agarose with a buffer consisting of 50 mM NaH2P04, pH 8.0, 300 mM NaCI, and 500 mM imidazole (buffer D). Twenty 0.5 rnL fractions were collected and kept on ice. Contents of the "pre-induction" control, "post- induction" control, "total-lysate" control, "flow-through" sample, "wash-through" sample, and selected eluted fractions were separated on a 12.5% polyacrylamide gel by SDS-PAGE (figure 1.15). EF-Tu protein was detected in the "post- induction'' control (lane c), "total lysate" control (lane d), and the eluted fractions
(lanes g - i), as expected. The EF-TU protein appeared > 95% pure after nickel- affinity chromatography. Seven eluted fractions containing' EF-Tu(NHis6) protein were pooled. The purified protein was dialyzed in a 2 L beaker against 1 L of buffer C with a stirring bar in the sliding-door fridge overnight. The next morning, a,, the dialyzed protein concentration was assayed using the B IO-RAD protein assay and by UV absorbance at 280 nm (molar extinction coefficient (E) = 41 600
M-'cm-' (102)). The protein was concentrated using a 4 mL ultrafree centrifuga1 filtration device equipped with a 5000 MW0porous membrane (Millipore,
Bedford, MA) with centrifugation at 4 OC and 4000 rpm in a Sorvall RT7 refrigerated centrifuge (Mandel Scientific Co., Ltd., Guelph, ON). After concentration, glycerol was added to a final v/v concentration of 50%. The protein was stored at a finai concentration of -53 pM in 400 pL aliquots at -20 OC.
1A3.2 Method 2 (resin-bound EF-TU)
Crude lysate was prepared as just described for method 1 (section
1.3.3.1) but the first dialysis step was omitted. The lysate was combined with 5 mL of Ni-NTA agarose (in ethanol) in a 50 mL polypropylene conical tube. The tube was capped and sealed with parafilm (to avoid leakage), and placed in a shaker in a 4 OC sliding-door fridge for 2 h, Two hours later, the mixture was poured into a 10 mL capped polypropylene column with a frit- The column was kept at 4 OC for 45 min undisturbed to allow the resin to settle at the bottom of the column. The colurnn was washed with 300 mL of buffer B and then 500 mL of a buffer containing 50 mM Tris-CI, pH 7.5, 30 mM KCI, and 10 rnM MgCl2 (buffer
E). Resin-bound EF-TU was aliquoted into 200 pL fractions and stored at 4 OC.
For EF-Tu concentration determination, one aliquot was washed with buffer D to elute the EF-Tu protein from the resin (vide infra: section 1-3.1 1-3 or 1.3.1 1.4).
The concentration of protein in the elution buffer was determined by UV absorbance at 280 nm (E = 41 600 ~-'crn" (102)). Each 200 pl aliquot contained -0.3 mg of protein- The elongation factor appeared > 95% pure as judged by SDS-PAGE analysis (data not shown).
1.3.4 Purification of E. coli EF-Tu(CHis6) from transformed JMl09 ceils
E. coli EF-Tu tagged with a C-terminal hexa-histidine group was purified from JMI 09 celis transformed with the corresponding plasmid following a modified version of a protocol reported by Boon and collaborators (124).
Glassware and LB media for cell cultures were autoclaved prior to use. Cells were obtained from a single isolated colony on a freshly-streaked plate that was made up of agar, LB medium, and 100 pg/mL ampicillin. A 20 mL culture containing LB medium, 100 pgfmL ampicillin, and JMI 09 cells transformed with plasrnid construct pKECAHis, in a 2 00 mL Erlenrneyer fiask, was incubated overnight in a 37 OC incubator shaker. The next morning, 1L of LB medium supplemented with 100 pgfmL ampicillin, in a 4 L Erlenmeyer flask, was incoculated with the 20 mL overnight culture. The large culture was allowed to grow in the 37 OC incubator shaker until an optical density of 0.8 at 600 nm was eached (-5 h). At this point, a 1 mL aliquot was removed and stored at 4 OC for
SDS-PAGE analysis ("pre-induction" control). The rest of the culture was induced with 1 mM freshly-prepared IPTG and allowed to grow for an additional
4.5 h. Four and a half hours later, another 1 mL aliquot was removed and stored at -20 OC for SDS-PAGE analysis ("post-induction" control). The cells were transferred to four 250 mL polypropylene copolymer centrifuge bottles and harvested by centrifugation at 4 OC and 7000 rpm for 20 min. Supernatants were
discarded by decantation while the cell pellets were stored in the bottles at -20
OC overnight. The next morning, the cells were thawed on ice (-1 h). The
thawed cells were combined and suspended in 20 mL of a buffer containing 50
mM Tris-CI, pH 7.6, 60 mM NH4CI, 7 mM MgCI2, 7 rnM PME, and 15% glycerol
(buffer F). The suspension was treated with freshly-prepared lysozyme (1
mglrnL) on ice for 30 min. The lysozyme-treated cell suspension was transferred
to two 50 mL polypropylene conical tubes. On ice, the cell suspensions were
sonicated. Sonication was carried out at half-maximal power with four 1 min
pulses at 30 s intervals. The sonicated cell suspensions were transferred to 50
rnL centrifuge bottles and centrifuged at 4 OC and 10 000 rpm for 30 min. The
supernatants were combined and transferred to a fresh 50 mL polypropylene
conical tube on ice. A 5 pL aliquot was removed and stored at -20 OC for SDS-
PAGE analysis ("total-lyçateJ'control). The crude lysate was combined with 5 mL
of Ni-NTA agarose (in ethanol). The tube was capped and sealed with parafilm, and placed on a shaker in a 4 OC sliding-door refrigerator for 2 h. After the
incubation, the mixture was loaded into a 1O mL capped polypropylene colurnn with a frit. The column was kept at 4 OC undisturbed for 45 min to allow the resin to settle at the bottom of the column. After sedimentation, the volume of liquid at the top of the column was allowed to pass through. From this flow-through, a 5 pL aliquot was removed and stored at 4 OC for SDS-PAGE analysis ("Row- through sample). The column was washed with a buffer containing 50 mM Tris-
CI, pH 8.0, 60 mM NH4CI, 7 mM MgC12, 7 mM PME, 15% glycerol, and 5 pM GDP (buffer G) to remove non-specifically bound protein and nucleic acid. Three hundred fifty millitre ofbuffer was required to remove ail non-specifically bound protein and nucleic acid and bring the absorbance at the two wavelengths to background values (OD = O - 0.02). From the wash, a 5 pL aliquot was removed and stored at -20 OC for SDS-PAGE analysis ("wash" sample). The pH of the column was then adjusted and equilibrated at 7.0 by washing with 50 mL of a buffer containing 50 mM Tris-CI, pH 7-0, 60 mM NH4Ct, 7 mM MgC12, 7 mM PME,
15% glycerol, and 5 PM GDP (buffer H). Nickel agarose-bound protein was eluted with a buffer that was made up of 50 mM Tris-Cl, pH 7-0,60 mM NH4CI. 7 mM MgC12, 7 mM PME, 15% glycerol, 80 mM imidazole, and 40 pM GD? (buffer
1). Forty fractions of 0.5 mL were collected and kept on ice. Contents of the
"pre-induction" control, "post-induction" control, "total-lysate" controt, "flow- through sample, "wash" sampIe, and selected eluted fractions were separated on a 12.5% polyacrylamide gel by SDS-PAGE (data not shown). The EF-Tu protein appeared > 95% pure after elution. Thirty-six of the fourty fractions collected contained EF-Tu(CHiss) and were pooled. The protein concentration was assayed using the BIO-RAD protein assay and by UV absorbance at 280 nm
(E = 41 600 ~%m-')(1 02). The protein was concentrated using an 4 mL ultrafree centrifugai filtration device equipped with a 5000 MWCO porous membrane with centrifugation at 4 OC and 4000 rpm in a refrigerated centrifuge.
The concentrated protein was stored in 50% glycerol at a concentration of -21 pM in 500 pL aliquots at -20 OC. 1.3.5 Aminoacylation of ~RNA'~'with L-cysteine
All tRNA manipulations were carried out wearing gloves. A ~RNA'~'stock
solution was prepared by dissolving 5 mg of lyophilized tRNA (Subriden RNA) in
200 pL of H20 in a 1.5 mL screw-cap microcentrifuge tube. The tube was
incubated in an 80 OC water bath for 3 min. Three minutes later, the tube was
placed in a regulated 37 OC water bath (Precision 180 series) for 20 min. This
heating-cooling cycle was carried out to denature the tRNA and allow proper
folding of the tertiary structure. Twenty min later, the tRNA concentration was
determined by UV absorbance at 260 nm, assuming that 1 unit = 2 nmol
(0.05 mg) tRNA (which corresponds to an approximate molar extinction
coefficient of 469880 ~"cm-'). The volume was adjusted to obtain a ~RNA'~'
stock with a concentration of -1000 FM. This ~RNA'" stock solution was stored
in a -20 OC freezer until required for aminoacylation experiments. When required,
the stock solution was thawed on ice (-7 min). After the amount required had
been withdrawn, the stock was immediately returned to the freezer.
~RNA~"was charged with L-cysteine by the partially purifid cysteinyl-
tRNA synthetase (section 1.3.1) in a 50 pL reaction which consisted of 50 mM
Tris-CI, pH 7-5,30 rnM KCI, 10 mM MgCl*, 5 mM DTT (dithiothreitol), 2 rnM ATP
(adenosine 5'-triphosphate), 4 mM cysteine, 100 pM tRNA, and 0.2 p.g/pL synthetase (scherne 1.3). DTT,ATP, and cysteine were aliquoted from stocks that were stored at -20 OC. The reaction was performed in a 1.5 mL microcentrifuge tube. For an aminoacylation reaction in which radiolabelled cysteine was used, the reaction was supplemented with 20 pCi of [35~]-cysteine E. coli CysRS (0.04 equiv,), DTT (50 equiv,), ATP (20 equiv,), 50 mM Tris, pH 7.5 (HCI), 30 mM KCI, 10 mM MgCl*
37 OC, 5 min (40 equiv.)
Scheme 1.3. Reaction scheme for preparation of Cys-tRNACYSfrom E. coli tRNACYS,L-cysteine, ATP, and E. coli CysRS. Menthe aa-tRNA forms, the amino acid is attached to the 2'-OH of the 3' terminal adenosine of the tRNA, obtained from a fresh stock solution with an approxirnate specific activity of 1000
Cilmmol. Radioactive cysteine was susceptible to oxidation; as a result, the corresponding stock solution was stored in a -80 OC freezer. When required for experiments, the stock was thawed at roorn temperature. After the amount required had been removed, the stock was immediately transferred to dry ice and subsequently a -80 OC freezer. As a control, an aminoacylation reaction in which active cysteinyl-tRNA synthetase was replaced by denatured CysRS was assembled as well. For convenience, the reaction containing active synthetase will be referred to as the "tesf' or "+Emreaction, while that containing denatured synthetase will be called the "controIn, "blank", or "-En reaction. The "test" and
"control" reactions were carried out in parallel. The reaction mixtures were assernbled on ice. The synthetase enzyme (denatured or active) was added last.
The reaction mixtures were incubated in a regulated 37 OC water bath for 5 min.
Five minutes later, the tubes were placed on ice. To each tube was immediately added 250 pL of H20 (to increase the volume) and then 300 pL of phenol (which had been equilibrated at pH 4.3 with 0.1 M citrate buffer). The tubes were mixed vigorously in an up-and-down fashion for 1 min at room temperature. Phenol denatures proteins (126); vigorous shaking was required to allow denaturation of the synthetase enzyme to happen efficiently. Removal of the synthetase protein was important so as to eliminate enzymatic deacylation (125). Also, the presence of protein would interfere with later steps. After phenol extraction, the tubes were centrifuged in a Micro 240A microcentrifuge (Denville Scientific Inc.) at rt (room ternperature) for 14 s at 13 000 rpm. From each sample, a 270 PL aliquot of the aqueous phase, which was the upper phase, was transferred to a fresh microcentrifuge tube at roorn temperature using an automatic pipettor. To precipitate tRNA, 30 pL of a solution containing 3 M NaOCOCH3 (pH 5.2.
(CH3COOH), and 900 pL of cold ethanol were then added (126). This step also separated tRNA from free cysteine and the other compounds from the original aminoacylation reaction mixture. The ethano[-containing solutions were placed in a -20 OC freezer for 30 min, then the precipitated tRNA was pelleted by centrifugation at 13 000 rpm for 15 min in a VSMC-13 microcentrifuge placed in a
4 OC sliding-door refrigerator. Supernatants were rernoved by decantation and pellets were washed with 75% ethanol (700 pL each). The pellets were air-dried for 2 min. They were each then redissolved in 50 pl of a buffer containing 1O mM NaOCOCH3, pH 4.5, and 5 mM tris(2-carboxyethyl)phosphine (TCEP) (buffer
J). Ethanol precipitation was repeated by addition of 220 pL of H20, 30 pL of 3 M
NaOCOCH3, pH 5.2, and 900 pL of ethanol. After incubation, centrifugation, and dissolution in the same manner as just described for the first round of tRNA precipitation, ethanol precipitation was repeated twice more, for a total of 4 ethanol precipitations. The 4 successive ethanol precipitations were required to completely remove free cysteine from tRNA. After the last ethanol precipitation step, C~S-~RNA~~was dissolved in 50 pL of buffer J.
An acidic buffer was mandatory to minimized chemical hydrolysis of the arninoacyl ester bond.
The role of TCEP in buffer J was to prevent disulphide bond formation between C~S-~RNA'" molecules via oxidation of the cysteinyl side chains by atmospheric oxygen. Disulphide-linked C~S-~RNA'" could be detected by urea-
PAGE (urea - polyacrylamide gel electrophoresis) with ethidium bromide staining
(vide infra: section 1-3.6). Typically < 10% disulphide-linked species were observed if the aminoacylated tRNA was stored in buffer J. If > 10% disulphide-
Iinked C~S-~RNA~~molecules were detected on an ethidum bromide-stained
urea-PAGE gel, the sample was incubated overnight (in bdfer J) (-15 h) at 4 OC prior to use in further experirnents.
Radiolabelled cysteine provided a means for calculating a yield for the aminoacylation reaction. Percentage aminoacylation was calculated by dividing the concentration of cysteine (as determined by liquid scintillation counting using an LS 60001C liquid scintillation wunter (BECKMAN)) by the concentration of tRNA (as deterrnined by UV absorbance at 260 nm).
[35~]-~ys-t~~~CFwas characterized by liquid scintillation counting, urea-
PAGE (on a 10% 8 M ureafrBE (TBE = Tris-borate/EDTA = 45 mM Tris-borate, pH 8.0, 1 mM EDTA) polyacrylamide gel) with ethidium brornide staining (vide infra: section 1.3.6), autoradiography (vide infra: section 1.3.7), and chemical hydrolysis (vide infra: section 1-3.9).
C~S-~RNA~"was either stored at -80 OC or used directly in a chemical modification experiment.
1.3.6 Urea-PAGE (with a 10% 8 M urealTBE polyacrylamide gel) with ethidium bromide staining for visualization of tRNA
To cast a 10% 8 M urea/TBE polyacrylamide gel, a 10 mL solution which consisted of 8 M urea, 1 X TBE, 10% acrylarnide, 0.5% bis-acrylamide, 112 pL of 10% ammonium persulphate (APS), and 5 pL of TEMED (N,N.N',Nt- tetramethylethylenediamine) was assembled in a 125 mL Erlenrneyer flask. A miniature slab gel (7 cm X 8 cm) was then cast using a BIO-RAD gel casting stand following the instructions in the ~ini-PROTEIN" II Electrophoresis Cell
Instruction Manual (810-RAD). The electrophoretic apparatus (~ini-PROTEIN" II
Electrophoretic Cell) with the gel incorporated was then set up according to BIO-
RAD'S instructions. Samples for loading were prepared by mixing an aliquot of a tRNA construct (unaminoacylated ~RNA'", C~S-~RNA~~,or chemically-rnodified
C~S-~RNA~~)(1 - 3 pL) with 1O pL of gel loading buffer (8 M urea, 0.02% bromophenol blue, 0.02% xylene cyanole, 1 X TBE). After samples were loaded
(using gel loading tips), electrophoresis was perforrned in 1 X TBE at a constant voltage of 300 V (power supplied by BIO-RAD'S PowerPac 300) for 10 min at room temperature. For ethidium bromide staining, the gel was imrnersed into a solution containing 40 pg/mL ethidium bromide for 5 min. Five minutes later, bands were visualized on a UV mini-transilluminator (120 VAC. 60 Hz, BIO-
RAD). Photographs of ethidium-bromide-stained gels were taken using a hand- held DS-34 Polaroid camera equipped with a deep yellow DS-34 camera filter
(B IO-RAD).
1.3.7 Autoradiography of 10% 8 M urealTBE polyacrylamide gels with bands that correspond to radiolabelled tRNA
Sarnples for analysis by autoradiography were first separated on a 10% 8
M urealTBE polyacrylamide gel by urea-PAGE as just described (section 1.3.6).
After electrophoresis, the gel was rinsed in H20for 15 min and then dried on a piece of ~hatman"filter paper using BIO-RAD'S model 543 gel drier. The dBed gel was then exposed on a phosphor screen in a cassette for 2 h. After the exposure, the bands were visualized by scanning the phosphor screen with a
Storm 860 phosphorimager (Molecular Dynamics). The scanner was controlled by a Scanner Control software (Molecular Dynamics). The image of the scan was analyzed using the Image Quant program (Molecular Dynamics).
1.3.8 Fluorescence imaging of fiuorescently-labelled CYS-~RNA~Y~
Sarnples for analysis by fluorescence imaging were first separated on a
10% 8 M ureaiTBE polyacrylamide gel by urea-PAGE as described in section
1-3.6. lmrnediately after electrophoresis, the gel was directly placed on a UV transilluminator. Fluorescence images were obtained using a digital camera
(RICOH RDC4200).
1.3.9 Chemical hydrolysis of the aminoacyl ester bond in cysteinyl-t~~~~~~
Chemical hydrolysis of [35~]-~ys-t~~~CySwas monitored at 4 different conditions: pH 4.514 OC, pH 8.314 OC, pH 45/22 OC (rt), pH 8.3122 OC, pH 7-3/37
OC, and pH 7.5/4 OC.
For a chernical hydrolysis experiment at pH 4.5 and 4 OC, [35~]-~ys-
~RNA~"was dissolved in 50 pL of a buffer that consisted of 5 mM NaOCOCH3. pH 4.5. and 5 mM DTT at a concentration of - 40 PM, in a 1.5 mL microcentrifuge tube. The solution was incubated in a 4 OC sliding-door refrigerator. Every hour, for a total of 4 hours, a 10 pL aliquot was transferred to a clean 1.5 mL microcentrifuge tube. The 10 pL aliquot was frozen on dry ice to prevent further deacylation. After the fourth aliquot had been removed from the cys-~RNA'" chemical hydroiysis reaction and frozen with dry ice, al1 aliquots were transferred to a -80 OC freezer for ovemight storage. The remainder of the original reaction mixture was allowed to incubate in the fridge ovemight. The next morning, a last 10 pL sample was transferred from the reaction mixture to a new microcentrifuge tube and frozen with dry ice. This aliquot corresponds to a
15 h time point. Ail 5 aiiquots were then thawed on ice (- 1 min). To each sample was added 260 pL of H20, 30 pL of a solution containing 3 M
NaOCOCH3, pH 5.2, and 900 pL of ethanol. These tRNA precipitations by ethanol served to remove free cysteine. The tubes were incubated at -20 OCfor
30 min. Thirty minutes later, they were centrifuged in a microcentrifuge that was equilibrated at 4 OC in a sliding-door refrigerator at 13 000 rpm for 15 min-
Supernatants were removed by decantation and each pellet was washed with
700 pL of a 75% ethanol solution. The pellets were air-dried for 2 min. The dried pellets were each dissolved in 10 pL of a buffer containing 5 rnM NaOCOCH3, pH
4.5, and 5 mM DTT. Each sample was assayed for percent aminoacylation by liquid scintillation counting and UV absorption at 260 nm (vide ultra: section
1.3.5). The percentage of intact [35~]-~ys-t~~~Cysat each time interval was plotted.
For a chemical hydrolysis experiment at pH 8.3 and 4 OC. [35~]-~ys-
~RNA~~'was dissolved at a concentration of -40 pM in 50 pL of a buffer containing 50 mM Tris-CI, pH 8.3, and 5 mM DTT in a 1.5 mL microcentrifuge tube. The sarnple was placed in the 4 OC sliding-door refrigerator. The first four
10 pL aliquots were transferred to clean microcentrifuge tubes, frozen on dry ice,
and subsequently transferred to the -80 OCfreezer at time intervals of 1 h, 2 h, 3
h, and 5 h. The remainder of the reaction mixture was allowed to incubate
overnight. The last 10 pL aliquot was withdrawn and frozen with dry ice at the
21 -5 h mark. AI1 five aliquots were thawed on ice. Subsequently, the samples
were treated in the same manner as just described for the deacylation
experiment at pH 4.514 OC (vide ultra). The percentage of intact [35~]-cysteinyl-
~RNA~~~at each time was plotted.
In the same manner as described for the deacylation experiment at pH 4.5
and 4 OC,a third chemical hydrolysis experiment at pH 4.5 and 22 OC was
performed. Aliquots of 10 pL were removed at time intervals of 1 h, 2 h, 3 h, 5 h,
and 20.25 h. The percentage of intact [35~]-~ys-t~~~CFat each time was
plotted.
A fourth chemical hydrolysis experiment at pH 8.3 and 22 OC was also
performed in the same manner as described for the deacylation experiment at pH
8.3 and 4 OC. Here, 10 pL aliquots were removed at time intervals of 1 h, 2 h, 3
h, 5 h, and 14.5 h. The percentage of intact [35~]-~ys-t~~~Cpat each time was
plotted.
For a deacylation experiment at pH 7.3 and 31 OC, [35~]-~ys-t~~~CFwas dissolved in 50 pL of a buffer containing 50 mM Tris-CI, pH 7.5, and 1 mM DTT in a 1.5 mL microcentrifuge tube. The 50 pL volume was divided into 5 equal aliquots of 10 pL each in 1.5 mL microcentrifuge tubes. Al1 five aliquots were placed in a regulated 31 OC water bath. At intervals of 10, 20, 30, 40, and 50
min, an aliquot was removed and frozen on dry ice. ARer al1 five aliquots had
been removed and frozen, the samples were thawed on ice. For each aliquot,
tRNA was precipitated with ethanol and dissolved in 10 pL of 5 mM NaOCOCH3,
pH 4.5, and 5 mM DIT in the same manner as just described for the other
deacylation experiments. The percentage of intact [35~]-~ys-t~~~CFat each
time interval was plotted.
In a sixth and final deacylation experiment, [35~]-cysteinyl-t~~~CYSwas
dissolved in a buffer containing 60 mM Tris-CI, pH 7.5, 60 mM NH&I, 60 mM
KCI, 12 mM MgC12, 12 mM PME, 20% glycerol, 1 mM GTP, and 10 pM GDP
which had been incubated in a 37 OC regulated water bath for 10 min and
subsequently cooled on ice for 2 min. All this was contained in a 1.5 mL
microcentrifuge tube. The concentration of arninoacylated tRNA in the sample
was 2 PM. A 100 pL aliquot was transferred to a new microcentrifuge tube which
was placed on ice. The rest of the reaction was placed in the 4 OC sliding-door
refrigerator. The 100 pL aliquot was transferred to a pre-chilled 4 rnL ultrafree
centrifuga1 filtration device equipped with a porous membrane with an MWCO of
5000. To the filtration column was added 4 mL of a buffer containing 100 mM
NaOCOCH3, pH 4.5 (buffer K). The colurnn was centrifuged for 5 min at 3800
rpm and 4 OC in a refrigerated centrifuge. After the centrifugation, another 4 mL of buffer L was added to the column. The column was centrifuged as just described. When the spin was finished, the sample retained above the membrane in the filtration unit was transferred to a clean 1.5 mL microcentrifuge tube which had been pre-chilied on ice. Percentage aminoacylation was calculated (vide ultra: section 1.3.5) by Iiquid scintillation counting and UV absorption at 260 nm. Aliquots of 100 pL were also temoved from the deacylation reaction mixture at time intervals of 15 min, 30 min, 45 min, 1 h, 1.25 h, 1-5 h, 1.75 h, 2 h, and 2.25 h. These aliquots were treated in the same manner as just described for the first 100 pL aiiquot Percentage of intact [35~]-
C~S-~RNA'"at each time point was plotted.
1.3.10 Chernical modification of C~S-~RNA~~~with [l-14~]-iodoacetamide, IAF, AIASS, and IAEDANS
The cysteinyl side-chain of ~~stein~1-t~~~~~~was alkylated with four different compounds: [1-14~]-iodoacetamide; 5-iodoacetarnidofluorescein (IAF)
(figure 1-1 Ga); 4-acetamido-4'-((iodoacetyI)arnino)stil bene-2'2-disulfonic acid
(AIASS) (figure 1. l6b); and 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1- sulfonic acid (IAEDANS) (figure 1.16~).The alkylation reaction was carried out using a procedure that was adapted from that developed by Kurzchalia and colleagues for labelling lysyl-t~~~LYS(L~S-~RNA~~~) (128) (scherne 1-4A). As controls, unacylated ~RNA'" was treated with each of the labelling reagents as well. Reactions with aminoacylated and unacylated tRNA were carried out in parallel for consistency in reaction conditions. The labelling reagent was first dissolved in 40 pL of dimethylsulphoxide (DMSO) (120 PL for reaction with IAF).
The DMSO was frozen on ice. Fifiy microlitre (150 pL for reaction with IAF) of
C~S-~RNA'~~(or unacylated tRNA) in 10 mM NaOCOCH3,pH 4.5,and 5 mM
TCEP was added to the frozen labelling reagent on ice. The sample was then
thawed by gentle mixing. After mixing, 10 pL (30 pL for reaction with IAF) of a
buffer containing 500 mM Tris-CI, pH 8.3 was added to initiate a reaction. The concentrations of the components in the reaction mixture were 50 mM Tris-Cl, pH
8.3, -20 pM C~S-~RNA~~~(-7 PM for reaction with IAF) (or unacylated tRNA),
40% DMSO, and -20 mM labelling reagent (1 1 mM at a specific activity 56 370 dpmfnmol for [l-14~]-iodoacetamide; 7 mM for IAF). After initiation, the reaction was incubated on ice and in darkness for 10 min- At this incubation time, no chemical hydrolysis of the aminoacyl ester bond of C~S-~RNA~~was detected at pH 8.3 and O OC (figure 1.19A). After the incubation, 170 pL of H20, 30 pL of a solution that consisted of 3 M NaOCOCH3, pH 4.5, and 900 pL of ethanol were added to precipitate the tRNA and remove unreacted labelling reagent, as well as the other small molecule components of the labelling reaction. The tube was placed in a -20 OC freezer for 30 min and then centrifuged at 4 OC for 15 min at a speed of 13 000 rpm. The supernatant was removed by decantation and the pellet was washed with 70G pL of a solution containing 75% ethanol. The pellet was air-dried for 2 min. The dried pellet was redissolved in 50 pl of the buffer that consisted of 10 mM NaOCOCH3, pH 4.5. An acidic pH was required to minimize chemical hydrolysis of the aminoacyl ester bond in the IabelIed Cys-
~RNA'". Ethanol precipitation was performed three more times in the same manner as just described. Each additional time, 220 gL of H20, 30 yL of the solution that contained 3 M NaOCOCH3, pH 5.2, and 900 pL of ethanol were added to the 50 plof tRNA. A total of 4 ethanol precipitations were required to completely remove unreacted labelling reagent. After the final ethanol precipitation, the chemically-modified ~~stein~l-t~~~~~~was dissolved and stored
in 50 pL of 10 mM NaOCOCH3, pH 4.5.
C~S-~RNA~"reacted with [l-14~]-iodoacetamide was characterized by
liquid scinitillation counting. Fluorescently-labelled C~S-~RNA~~~constructs were characterized by fluorescence spectroscopy using a Perkin Elmer LS-5OB
luminescence spectrophotometer and a cuvette with a 0.5 cm path length and fluorescence imaging on a UV mini transillurninator of a 10% 8 M ureaKBE polyacrylamide gel. For the fluorescence ernission scans, the excitation wavelengths for the different fluorophores are specified in the legend of figure
1.22.
A product yield was deterrnined for the labelling reaction with [l-14c]- iodoacetamide. The amount of 14~-labelledacetamido-C~S-~RNA'~
(iodoacetamide-labelled C~S-~RNA~~~)produced was determined from liquid scintillation counting. To determine the fraction of ~~steinyl-t~~~~~that was converted to the labelled counterpart, the arnount of labelled tRNA was divided by the original amount of unlabelled C~S-~RNA~~(as determined from a separate aminoacylation experirnent with [35~]-cysteine).
The chemically-modified cys-t~~ACYJconstructs were either stored in a -
80 OC freezer or used directly in EF-Tu binding experirnents.
1.3.1 1 Ternary cornplex formation and detection
Ternary complex formation between T. thermophilus EF-Tu - both in the
GDP- and GTP-bound states - and arninoacylated tRNA constructs rS~]-~ys-
~RNA'" ([35~]-~ys-t~~~C~-~~-~u-~~~~~~~),IAF-C~S-~RNA~~( IAF-labelled C~S-~RNA~~)(IAF-C~S-~RNA~~~-EF-TU-GTP/GD P), AIASS-C~S-~RNA~~~ (AIASS-
labelled cys-~RNA'~)(AIASS-C~S-~RNA~~~-EF-TU-GTPIGDP), and IAEDANS-
C~S-~RNA~~(IAEDANS-labelled C~S-~RNA'~)(IAEDANS-C~S-~RNA~~-EF-TU-
GTPIGDP) were monitored by method 1 and method 2 (vide infra: sections
1.3.1 1.1 and 1-3.1 1.2, respectively), both of which are rnodified-versions of a
protocol published by Ribeiro and colleagues (105) for the purification of E. coli
~RNA'~~and T. thermophilus ~RNA~~'from crude tRNA mixtures. Binding of
[35~]-~ys-t~~~CFto E. coli GDP- and GTP-bound EF-TU, and [14~]-labelled
acetarnido-~~s-t~~~~~~to T. thermophilus GDP- and GTP-bound EF-Tu
(~C~~~~~~~-C~S-~RNA~~-EF-TU-GTPGDP)were assayed using just method 1.
1.3.1 1.1 Method 1 -- EF-TU-GDP binding assay
In a 1.5 mL microcentrifuge tube sitting on ice, a 1 mlreaction mixture that consisted of EF-Tu-GDP (aliquoted directly from a stock (vide ultra: section
1-3.3.1 )) (concentration specified in the legends for figures 1-23 - 1.28), 50 mM
Tris-CI, pH 7.5, 50 mM KCI, 50 mM NH&I, 10 mM MgCI2, and 10 mM PME was assembled. To the mix was added a freshly-thawed 50 pL aliquot of an aminoacylated tRNA construct (concentration specified in the legends for figures
1.23 - 1.28) in 10 mM NaOCOCH3, pH 4.5 (and 5 mM TCEP for experiments with
[35~]-~ys-t~~~CF).The mixture was incubated on ice for 2 min. Two minutes later, it was combined with 0.5 mL of Ni-NTA agarose (in ethanol) in a 15 mL polypropylene tube. The tube was capped and sealed with parafilm (to avoid teakage) and placed on a shaker- in a 4 OC sliding-door refrigerator for 10 min.
Ten minutes later, the nickel-agarose mixture was loaded into an empty capped 2 mL polystryene column with a frit (Pierce, Rockford, IL). The column was
allowed to stand in the fridge undisturbed for 30 min to allow the resin to
completely settte at the bottom of the column. After complete sedimentation was
observed, the volume of iiquid at the top of the Ni-NTAIagarose resin was
allowed to pass through and collected in a single 1.5 mL microcentrifuge tube
("flow-through" sample). This flow-through sample was stored at 4 OC. The
column was washed with 10 mL of a buffer containing 50 mM Tris-CI, pH 7.5, 50
rnM NH&I, 50 mM KCI, 10 mM MgCI2, 1 mM GTP, and 5 rnM PME (buffer L).
One millilitre fractions were collected in 1.5 mL microcentrifuge tubes and stored
at 4 OC- The column was washed a second time with 1O mL of a buffer
containing 50 mM Hepes (N-2-hydroxyethylpiperazine-NI-2-ethanesulfonic acid),
pH 7.5, 150 mM NaCI, 50 mM NH4CI, IOmM MgCI2, 5 mM PME, and 50 pM GTP
(buffer M). Again, 1 mlfractions were collected in 1.5 mL microcentrifuge tubes
and stored at 4 OC. Ternary complexes of aa-tRNA-EF-Tu-GTP were eluted from
the nickel-affinity column with 5 mL of a buffer containing 50 mM NaH2P04at pH
8.0 (NaOH), 300 mM NaCI, and 500 mM imidazole (fl uorimetric grade imidazole
was used for experiments with AIASS- and IAEDANS-modified C~S-~RNA~~~
constructs). Fractions of 0.5 mL (0.8 mL for experiments with fluorescently-
labelled C~S-~RNA~~constructs) were collected in 1.5 mL microcentrifuge tubes
and kept at 4 OC.
For experirnents involving [35~]-~ys-t~~~CFand [l-14~]-iodoacetamide-
modified C~S-~RNA~",the radioactivity in each fraction was measured by liquid scintillation counting of a 100 pL aliquot dissolved by 10 mL of liquid scintillation cocktail (ACS). For experiments with fiuorescently-labelled C~S-~RNA'" constructs, the fluorescence in each fraction was monitored by fluorescence spectroscopy. An elution profile was generated from each experiment.
1.XI 1.2 Method 1 -- EF-TU-GTP binding assay
In a 1-5 ML microcentrifuge tube sitting on ice, a 1 mL reaction mixture. that consisted of EF-Tu-GDP (aliquoted directly from a stock (vide ultra: section
1.3.3.1 ) (concentration specified in the legends for figures 1.23 - 1.28), 5 mM phosphoenolpyruvate (PEP), 1 mM GTP, 50 mM Tris-CI, pH 7.5, 50 mM KCI, 50 mM NH4CI, 10 mM MgCl2, 10 mM PME, and 100 pgImL pyruvate kinase (PK) was assembled. The reaction mixture was incubated in a regulated 37 OC water bath for 10 min. The assay was a nucleotide exchange reaction in which EF-Tu-
GDP was converted to EF-Tu-GTP. After the incubation, the mixture was placed on ice. The rest of the procedure is identical to the protocol just desctibed for the
EF-Tu-GDP binding assay (vide ultra: section 1-3.1 1 -1 ), starting with the addition of a freshly-thawed 50 pl aliquot of an aminoacylated tRNA construct
(concentration specified in the legends for figures 1-23- 1.28) in 1O mM
NaOCOCH3, pH 4.5 (and 5 mM TCEP for experiments with [35~]-~ys-t~~~Cp).
1.3.1 1.3 Method 2 -- EF-TU-GDP binding assay
A 400 pL reaction consisting of 200 pL of Ni-NTAIagarose resin-bound
EF-Tu (aliquoted directed from a stock (vide ultra: section 1.3.3.2)) (-0.3 mg protein) and 3 mM GDP was prepared on ice (in triplicates) in 1.5 mL microcentrifuge tubes. To these tubes were added aliquots of an aminoacylated tRNA construct. For expenments involving [35~]-~ys-t~~~CFand IAF-labelled
C~S-~RNA~~,there was -0.5 nmol of aminoacyl tRNA in the binding assays. For
experiments with AIASS-derivatized and IAEDANS-modified C~S-~RNA'"
constructs, there was -2 nmol of aa-tRNA in the binding assays. The reaction
mixtures were vortexed gently at room temperature for 30 s and then added to
empty capped and pre-chilled 2 mL polystyrene colurnns with frits. The caps
were rernoved and the columns were placed in 50 mL polypropylene conical
tubes. The tubes were capped and centrifuged for 1 min at 1000 rpm and 4 OC.
After the centrifugation, a 300 pL aliquot of a buffer containing 50 mM Tris-CI, pH
7.5, 30 mM KCI, and 1O mM MgCI2 (buffer E) was added to each colurnn. The
columns were centrifuged using the conditions just described. The columns were
then washed a second and third time in the same manner with 300 pL and 400
pl, respectively, of buffer E. The flow-throughs were transferred to 1.5 mL
microcentrifuge tubes ("flow-through" fractions), which were stored at 4 OC. A 3
mlaliquot of buffer E was then added to each column. The tubes were
centrifuged for 5 min at 1000 rpm and 4 OC. Five minutes later, the eluates were transferred to 1.5 mL microcentrifuge tubes, which were stored at 4 OC ("washJ1 fractions). For eluting ternary complexes of aa-tRNA-EF-Tu-GTP from the Ni-
NTA agarose, 500 pL of a buffer containing 50 mM NaH2P04, pH 8.0, 300 mM
NaCI, and 500 mM fluorimetric grade imidazole (buffer D) was added to each column. The tubes were centrifuged at 1000 rpm and 4 OC for 1 min. The elution step was repeated a second and third time with 500 pL and 1 mL, respectively, of buffer D (containing fluorimetric grade imidazole) in the same manner as just described. The elutates were transferred to 1.5 rnL microcentrifuge tubes, which
were stored at 4 OC ("elution" fractions).
For experiments with [35~]-cysteinyl-t~~~C~the radi oactivity in the "flow-
through", "wash", and "elution" fractons was detected by Iiquid scinitillation
counting of a 100 pL aliquot dissolved in 10 mL of liquid scintillation cocktail. The
results were pIotted,
For experiments involving fluorescently-labelled C~S-~RNA~~~constructs,
the "elution" fractions were transferred to 4 mluitrafree centrifuga1 filtration
devices equipped with a membrane with an MWCO of 5000, The filtration units were centrifuged at 3200 rpm for 30 min at 4 OC, after which, the volume of
sampIe retained above the membrane was transferred to a eesh filtration unit.
After addition of 4 mlof the 50 rnM Tris-CI, pH 7.5, 30 rnM KI, i O mM MgCI2
buffer (buffer E), each column was centrifuged again. The retained samples were transferred to a third set of new filtration devices. Four millilitres of buffer E was added to each column. The columns were centrifuged ai 3200 rpm and 4 OC for 30 min. The samples in the column were transferred to 1-5 mL microcentrifuge tubes. The volumes were adjusted to 9 50 pL with buffer E.
Fluorescence detection in the "flow-through", " wash", and concentrated "elutionJJ sampies was determined by fluorescence spectroscopy using a cuvette with a
0.5 cm path length. The excitation wavelengths for the different Ruorophores are specified in the Iegend of figure 1.22. 1.3.11.4 Method 2 -- EF-Tu-GTP binding assay
A 400 pL reaction that consisted of 200 pL of Ni-NTNagarose resin-bound
EF-Tu (afiquoted directly from a stock (vide ultra: section 1.3.3-2)) (-0.3 mg
protein), 3 mM GTP, 12 mM PEP, and 0.02 pg/pL pynivate kinase (2 units of
activity (Sigma)) was prepared on ice (in triplicates) in 1.5 mimicrocentrifuge tubes. These reaction mixtures were incubated in a regulated 37 OCwater bath for 1.5 h and then placed in a 4 OC sliding-door fridge for 30 min. The rest of the procedure is identical to the protocol just described for the EF-Tu-GDP binding assay (vide ultra: section 1.3.1 1.3), starting with the addition of an aliquot of an aminoacylated tRNA construct.
For both method 1 and method 2, EF-Tu-GDP and EF-Tu-GTP binding assays for an aminoacylated tRNA construct were carried out sirnultaneously.
1.XI 2 Chernical hydrolysis of EF-Tu-GTP-protected CYS-~RNA~Y=
A 2 mL sample of EF-Tu-GTP-protected [35~]-~ys-t~~~CFin a buffer containing 50 mM NaH2P04,pH 8.0, 300 mM NaCI, and 500 mM imidazole, in a
15 mL polypropylene tube, was prepared using method 2 (vide ultra: section
1.3.1 1.4). For a chernical hydrolysis experiment, it was stored in a 4 OC sliding- door refrigerator. From the stock, a 100 pL aliquot was transferred to a 4 mL ultrafree centrifuga1 filtration device equipped with a membrane with a molecular weight cut-off of 5000. The column was centrifuged for 10 min at 3700 rpm and
4 OC. After the centrifugation, 1 mL of a buffer consisting of 10 mM N~OCOCHJ, pH 4.5 (CHJCOOH) was added to the column. The filtration unit was centrifuged at 3700 rpm and 4 OC for 20 min. The purpose of the wash was to remove free
radiolabelled cysteine. The sample retained above the membrane was combined with 10 mL of liquid scintillation cocktail (ACS) and assayed for total amount of
radioactivity (which corresponded to radioactive ternary complexes). Aliquots of
100 pL were removed from the original [35~]-~ys-t~~~C~~~-~u-~~~stock kept at 4 OC at time intehals of 1 h, 2 h, 3 h, 4 h, 5 h, 7 hr, and18.25 hl and treated in the same manner as just described for the first 100 plaliquot. The radioactivity detected at each time point was plotted.
Using the same procedure as just described, deacylation experiments for
[35~]-~ys-t~~~Cp-~~-~~-~~~were also performed at 24 OC (rt) and 31 OC. All other conditions were the same. Here, aliquots were examined at time intervals of 0.5, 1, 1.5, 2, and 2.5 h. For each experiment, the radioactivity detected at each time interval was plotted.
1.XI 3 Molecular rnodelling
1.XI 3.1 Structure of the binding site cavity
The structures of C~S-~RNA~~~-EF-TU-GDPNPand P~~-~RNA~"-EF-TU-
GDPNP were obtained from the RSCB protein data bank (entry codes 1B23 and lm,respectively). Binding pockets were defined using the program SURFNET
(129) and visualized using GRASP (130) running on a Silicon Graphics SGI
Octane RI0000 computer. A subset of residues was defined in which at least one atorn of each residue in the subset was within 15 A of an atom of the amino acid esterifîed to the 3' nucleotide. This subset was large enough to encompass the entire binding pocket and a majority of the EF-Tu structure but excluded al1 but the acceptor stem of the tRNA. Hydrogens were added to the structure, water mo~eculeswere removed, and the amino acid (Phe in UïT and Cys in
1823) was removed using the program HYPERCHEM (Hypercube, Gainesville,
FL). The resulting coordinate file was used as input for the program SURFNET which produces a file describing the size and shape of any cavities in the structure. A grid separation of 0.8,a minimum radius for gap speres of 1.O, and a maximum radius for gap spheres of 4.0 was used. The cavity corresponding tu the amino acid binding pocket was located by inspection of the cavity map produced by SURFNET superimposed on the input structure including the amino acid esterified to the 3' nucleotide. A coordinate file defining the structure of the binding pocket only was then produced using the MASK routine within
SURFNET.
The surface of the ternary compiex (with hydrogens, without water, and without the amino acid esterified to the 3' nucleotide) was rendered using the program GRASP. A rnolecular surface representation of the binding pocket only was then obtained by deleting al1 parts of the surface that were more than 2.0 A from the coordinates of the binding pocket as determined by SURF \1 ET.
1.3.1 3.2 Structure of the chemically-modified amino acid
The structure of fluorescein-labelled C~S-~RNA'"was mode led starting from the reported structure of the ternary complex (1823). The structure of fluorescein (figure 1-16a) was drawn and minimized using the HYPERCHEM implementation of the MM3 molecular mechanics package. The fluorescein group was then docked interactively with the ternary complex within
HYPERCHEM. The structure was modified so as to chemically join the fluorescein group to the cysteine side-chah (structure in figure 1.16a) and conformational energy was minimized, allowing onty the fluorescein group and the cysteine side chain to move. One thousand cycles of minimization by the method of steepest descents fotlowed by minimization to convergence (rms gradient c 0.1 kcai/(Amoi)) using the Polak-Ribiere algorithm was performed.
The MM3 force field was employed with the bond dipoles option and no cutoffs. 1.4 Results and Discussion
1.4.1 Synthesis of cy~tein~l-t~~~~~~
Purified E. coli ~RNA'" was aminoacylated with L-cysteine in an enzymatic reaction using partially purified E. coli cysteinyl-tRNA synthetase as the catalyst and AT? as a cofactor. The scheme is shown scheme 1.3.
The amount of synthetase used in the aminoacylation reaction was detennined from an independent experiment in which a series of arninoacylation reactions of the type shown in scheme 1.3, each containing a different level of synthetase, with radiolabelled cysteine were carried out. The graph in figure
1.1TA shows that 10 - 60 pg of synthetase gives rise to the sarne amount of aminoacylated tRNA.
In a different experiment, the incubation time, rather than the level of cysteinyl-tRNA synthetase, was varied. The graph in figure 1 -178 reveals that the level of aminoacylation is the sarne whether the reaction is carried out for 5,
10, 15, 20, 25, or 30 min.
VVith the conditions shown in scheme 1.3, a maximum level of cysteine incorporation of approxirnately 1300 pmol of cysteine per unit of tRNA was observed. This corresponds to approximately 40% of the original amount of tRNA being aminoacylated. This is similar to values reported in the literature
(34)-
The incomplete conversion of ~RNA'~'to the aminoacylated form would be explained if a fraction of the tRNA was somehow not recognized by the synthetase. It has been reported that the E. coli cysteine-specific tRNA has an Quantity of sy nthetase (microgram)
Tirne (minute)
Figure 1.17. A Iine graph for production of Cys-tRNACYS(using scheme 1.3) using different arnounts of cysteinyl-tRNA synthetase (A) and at different incubation times (B). unusual metabolic property in that it is subject to rapid end-turnover (i-e. the
terminal adenosine residue becornes detached from the rest of the tRNA) (75).
Without the terminal A, ~RNA'" would beunable to accept the cysteine amino
acid during the aminoacylation reaction. The Subriden source of ~RNA'" may
contain a fraction of this defective entity. The degradation can only be reversed with the assistance of tRNA nucleotidyltransferase.
For reactions in which 35~-labelledcysteine rather than cold cysteine was employed, freshly prepared [35~]-cysteinealways gave higher levels of
incorporation presumably because oxidation of cysteine to cystine or cysteic acid occurred in the radioactive stock solution over time. This trend was evident from
21 independent experiments in which radioactive cysteine stocks with different preparation dates were used. The lowest level of charging detected was 5%, which was from an experiment using [35~]-cysteinethat was 1 month old.
It has been reported that the cysteinyl-tRNA synthetase aminoacyIates
~RNA~"with cysteine at either the 2'-OH or 3'-OH (of the terminal adenosine residue) attachment site (131 ). However, the mechanistic details of the reaction remain to be determined. Crystals of the E. coli CysRS suitable for high- resolution structure determination have reportedly been obtained (132).
1.4.2 Characterization of cysteinyl-t~~~~~~
~RNA'" aminoacylated with [3s~]-cysteinewas characterized by liquid scintillation counting, autoradiography, urea-PAGE with ethidium bromide staining, and chemical hydrolysis at alkaline pH (vide infra: section 1.4.4). From a 50 pL sample containing -40 pM radiolabeiled C~S-~RNA~~(-222
000 dpmfnmol) (dpm = disintegration per minute) dissolved in 10 mM
NaOCOCH3, pH 4.5 (CH3COOH), which, for simplicity, will sometimes be referred to as the "test" or "+Ensample, a 1 pL aliquot typically gave a 12 000 cpm (counts per minute). An aliquot of the same voiume containing an equivalent amount of unacylated tRNA that had been treated with [35~]-cysteine
(also -222 000 dpmlnmol) and denatured cysteinyl-tRNA synthetase, which, for simplicity, will sometirnes be referred to as the "control" or "-E sample, typically produced a count of 500 cpm. Background was typically 30 cpm. The huge difference in radioactivity between the "blank" and "test" samples (1 1 500 cpm) provided evidence for a successful aminoacylation reaction between cysteine and ~RNA'~' as catalyzed by cysteinyl-tRNA synthetase with ATP as a cofactor.
A 1 pL aliquot from each of the "test1'and "blank" samples was analyzed by urea-PAGE with ethidium bromide staining (figure 1.1 8A). From urea-PAGE, two distinct bands were observed for ~RNA'" treated with [35~]-cysteine,ATP, and native CysRS (lane a) whereas one band was detected for ~RNA~~treated with [35~]-cysteine,ATP, and denatured CysRS (lane b). For the latter situation, the band presumably corresponds to unaminoacylated tRNA. The identity of the bands for the former case was uncertain and therefore further experiments were required for determination. The lower band is presumably partly from unaminoacylated tRNA.
To deterrnine the identities of the two bands for the "test" sample, a 1 pL aliquot from each of the "+El1and "-E samples was analyzed by autoradiography disulphide-linked disulphide-linked CYS-tRNACYs [35S]-Cy~-tRNACYs unaminoacylated tRNACYsand CptRNACYS [35S]-Cys-tRNACYS (monomer) (monomer)
Figure 1.18. (A) Polaroid picture of an ethidium bromide-stained 10% 8 M urea/TBE polyacrylamide gel. Lane a: tRNACYstreated with [35S]-cysteine,ATP, and native CysRS; and lane b: ~RNACYS treated with [35S]-cysteine,ATP, and denatured CysRS. Aliquots of 1 pL were loaded (section 1.35). (0) Autoradiograph of (A). as well (figure 1.1 88). From autoradiography, two distinct bands (of nearly equal
intensity) were again observed for ~RNA'~treated with [35~]-cysteine,ATP, and
native CysRS (lane a) while no band was detected for ~RNA'" treated with [35~]-
cysteine, ATP, and denatured CysRS (lane b), as expected. The two radioactive
bands for ~RNA'" treated with radioiabeiled cysteine, ATP, and native CysRS
have the sarne mobilities as the two bands from the urea-PAGE experiment.
They were tentatively identified as signals from [35~]-~ys-t~~~C"(lower band)
and disulphide-linked [35~]-~ys-t~~~CyS(upper band).
To check this hypothesis, the "test" sample was then treated with 5 mM tris(2-carboxyethy1)phosphine (a reducing agent) ovemight (-1 5 h) at 4 OC.
Chernical hydrolysis of the aminoacyl ester bond in C~S-~RNA'~~was not
observed under these conditions (figure 1.19A). One microlitre of this TCEP- treated sample was analyzed by urea-PAGE with ethidiurn bromide staining (data not shown) and autoradiography (figure 1.21, lane b). In both assays, only one band with the sarne electrophoretic mobility as that observed for unaminoacylated tRNA (cf. figures 1.1 8A (lane b) and 1.21 (lane b)) was detected. Comparing these results with the urea-PAGE and autoradiography results for the "+EVsample untreated with TCEP revealed that TCEP treatment caused a disappearance of the upper band and an increase in the intensity of the lower band (cf. figure 1.21, lanes a and b). The TCEP-treatment experiment confirmed that reduced and disulphide-linked C~S-~RNA~"have different mobilities on a 10% 8M urealTBE polyacrylamide gel with the slower migrating band corresponding to the disulphide-linked species. The oxidation of cysteinyl- ~RNA'~'to disulphide-linked C~S-~RNA~~~presumably arose from reaction with atmospheric rnoiecular oxygen.
For practical purposes, such as chernical modification of the cysteinyl side-chain, samples containing C~S-~RNA'~constructs were always dissolved and stored in buffers containing 5 mM freshly-prepared TCEP. Old solutions of
TCEP (> 1 week) were not as effective as freshly-prepared TCEP in reducing oxidized ~~stein~l-t~~~~~.C~S-~RNA~~kept in TCEP-containing buffers typically contained c 10% disulphide-linked species, as judged by urea-PAGE with ethidium bromide staining. Sarnples in which > 10% was detected were incubated for 15 h at 4 OC in fresh TCEP.
More evidence for the attachment of cysteine to ~RNA'" via an aminoacyl ester linkage in C~S-~RNA~~came from experiments which showed that the cysteinyl group was quickly cleaved from the terninal adenosine residue of
~RNA'~'upon incubation in rnildly alkaline solution (pH 8.0) (vide infra: section
1.4.4).
1.4.3 Cornparison between DTT and TCEP in terms of their abilities to red uce disulphide-linked C~S-~RNA~~'
DTT and TCEP are two of the most commonly used reducing agents in biological experiments. While treatment of disulphide-linked C~S-~RNA~~in the presence of TCEP for 15 h at pH 4.5 and 4 OC yielded -100% C~S-~RNA~~* monorners (cf. figure 1.21, lanes a and b), DTT appeared to be only effective at a pH > 8 and a temperature > 4 OC (data not shown). This result is consistent with the report that TCEP is capable of reducing disulphide-containing compounds rapidly and completely under very acidic conditions, even in the presence of 100 mM HCI, pH 1.5 (133), whereas DTT becornes inactive at low pH (134). In fact, below pH 8, TCEP is more effective than DlTfor reduction of disulphide bonds
(133). This feature is important as deacylation of ~ys-~RNA'"is relatively rapid at pH 8 and above (vide infra: section 1.4.4). Getz and colleagues have shown that TCEP is more stable than DTT during long-term storage in soIution (135).
This is significant because TCEP was required for the storage of the C~S-~RNA~~~ preparations. TCEP does not appear to react easily with iodoacetamide- or maleimide-containing compounds (135), whereas dithiothreitol cornpetes with other thiol-containing compounds for chemical modification (1 35, 136). To compensate for reaction with DTT, a larger excess of Iabelling reagent is required. Two other reasons that TCEP is an ideal reducing agent for disulphide- containing compounds are that it is highly water-solubility (310 g/L), highly selective for disulphide bonds, and unreactive toward other functional groups commonly found in proteins (137, 138).
1.4.4 Stability of the aminoacyl ester bond in C~S-~RNA~~~
The stability of the aminoacyl ester bond in ~~stein~l-t~~~~~toward hydroxide-catalyzed hydrolysis was a critical factor in our investigations. Intact
C~S-~RNA'"was required for synthesis of fluorescent1y-labelled ~ ys-~RNA'? In order to deterrnine the appropriate conditions for long-term storage as well as labelling conditions that would minimize the extent of deacyiation while the cysteinyl side chain was being derivatized, it was important to know the susceptibility of the aminoacyl ester bond at different pH values and temperatures.
Frorn the chemical hydrolysis experiments, a series of half-lives as a function of pH and temperature for the aminoacyl ester bond of C~S-~RNA'" were determined (table i-1 and figure 1-1 9). At 4 OC, the half-life (tli2) of cysteinyl-~RNA'" was obsewed to be -1 5 h at pH 4.5 (figure 1.19A) but only -2 h at pH 7.5 (graph not shown) and -2 h at pH 8.3 (figures 1.1 9A)- At 22 OC (rt), the tins were -20 h at pH 4.5 and -1.25 h at pH 8.3 (figure 1-19B). At 31 OC,the half-life was -0.5 h at pH 7.3 (figure 1.19C). Essentially, C~S-~RNA~~'is stable at acidic pH and low temperature, and susceptible to chemical hydrolysis at alkaline pH and high temperature.
The stability of the arninoacyl ester bond in C~S-~RNA~~has not been extensively discussed in the Iiterature. The only report is from Hentzen who studied the rate of hydrolysis in a buffer containing 0.1 M Tris-CI, pH 8.6, at 37 OC
(139). The reported half-life is 16 min (139), which is in Iine with our results. In his work, Hentzen also looked at the stability of the arninoacyl ester bond in 18 other E. coii aminoacyl-tRNAs. The aa-tRNAs, together with their respective half-lives at pH 8.6 and 37 OC, are Iisted in table 1.2. The relative order for proline, leucine, histidine, phenylalanine, threonine, and valine were observed by other investigators as well (88, 89, 140, 141). Table 1.1. Half-lives for the aminoacyl ester bond in ~ystein~l-t~~~~~ at different pHs, temperatures, and buffer compositions.
Buffer PH Temperature (OC) Half-life (h)
5 mM NaOCOCH3, 4.5 4 -1 5 5 mM DTT
50 mM Tris-Ci, 8.3 5 mM DTT
5 mM NaOCOCH3, 4.5 5 rnM DTT
50 rnM Tris-Cl, 8.3 5 mM DTT
50 mM Tris-Cl, 7.3 1 mM DTT
60 mM Tris-CI, 7.5 60 mM NH4CI, 60 mM KCI, 12 mM MgCI2, 12 mM PME, 20% glycerol, 1 mM GTP, 10 uM GDP
Table 1.2. Reported half-lives for 19 natural aminoacyl-tRNAs at pH 8.6 and 37 OC. (Data obtained from Hentzen paper (139)).
Aminoacyl-tRNA Half-life (min) Aminoacyl-tRNA Haff-life (min)
P~O-~RNA~'~ 2 ~~s-tRNA~~' 14
la-t RNA~'~ 6 T~~-~RNA~~15
L~u-~RNA~'" 7 H~s-~RNA~" 16
GIY-~RNA~" 8 C~S-~RNA~~~16
GIU-~RNA~'" 9 Phe-t~~~'~~ 16
G ln-~RNA~'" 9 ser-t~~~~~'17
ASP-tRNA~~P 11 ~hr-tRNA~~' 38
ASn-t R NA^'" ? 1 va1-t~~~~~'60
Arg-t RNA~'~ 12 Il e-t RNA"" 65
et-t RNA~" 12
The variation in aminoacyl ester bond stability for the 19 aminoacyl-tRNAs appears to be a reflection of the different natures of the side-chains. This was suggested by experiments frorn Strickland and colleagues who studied the effect of side chain structure on the rate of hydrolysis of aa-tRNA (141). The investigators observed that ~al~1-t~~~~~~and phenylalanyl-~RNA'~~(two aa- tRNAs which differ chemically only in the aminoacyl side chain) exhibited different rates of hydrolysis (tln = 318 min and 69 min, respectively). Likewise,
~RNA"~'charged with valine or phenylalanine exhibited a half-life of 324 min and
81 min, respectively. Transfer RNA structure had no significant effect on the stability of the aa-tRNAs since deacylation rates of va1-t~~~~~'and va1-t~~~'~~ were similar, as were the deacylation rates of P~~-~RNA'",and P~~-~RNA"~'
(142).
It appears that the differences in the rates of deacylation for the 19 aa- tRNAs are caused by different degrees of inductive effect exhibited by the aminoacyl side-chain groups; that is, the arninoacyl-tRNA with the more powerful electron-withdrawing side châin exhibits the faster rate of hydrolysis. This was suggested by Schuber and collaborators who studied the sensitivity of hydrolysis as a function of the nature of the aminoacyl side chain (90). They plotted the rate of hydrolysis versus the equilibrium acid dissociation constant (pK) for the amino acid acidic function for a series of aa-tRNAs (C~S-~RNA~~~,P~~-~RNA'~~, Met-
~RNA~~',ser-t~~~'~~, pro-~RNA'", T~~-~RNA~~',GI~-~RNA~'~, GIU-~RNA~", Leu-
~RNA~~',A~~-~RNA*'~, V~I-~RNA~~', and lle-~RNA"~) (90). The p& of the arnino acid carboxylic acid provides a measure of the inductive effect of the side chain
(e-g- the stronger the acid, the greater the withdrawal of electron density from the carboxyl group by the side chain). The researchers observed that deacylation rates were higher for the stronger acids (Le. side chains with stronger electron withdrawing groups), and a trend that was similar to that displayed in table 1.2
(90). This implies that the cysteinyl side chain withdraws less (or donates more) electron density from the a-carbonyl rnoiety than do the side chains from the amino acids listed before cysteine (from Ala to Met) in table 1.2; exhibits a similar inductive effect as the lysyl, tyrosyi, and histidyl side chains; and withdraws more electron density than do the aminoacyl side chains listed after the cysteinyl side chain (except for the valyl and isoleucyl side groups). For valine and isoleucine, the stability of the aminoacyl ester bond is infiuenced by steric effects as well (as observed by Schuber and CO-workers)(90). P~O-~RNA~~~,on the other hand. is characterized with an amino group that is more basic (pK = 8.7) (90) than the amino groups of the other aa-tRNAs (which are typically between 6.7 and 7.8)
(142), and is therefore the most sensitive to chernical deacylation (vide infra).
Hay and colleagues measured a kbs(pseudo first order rate constant) for hydrolysis of methyl S-methylcysteinate (Le. methyl ester of C~S-~RNA~"in which the thiol group is methylated) at pH 11-0 and 25 OC (142). The value reported is -0.08 min-' (which corresponds to a tln of -9 min) (142), which corresponds to a rate that is significantly slower than the rate of deacylation of cysteinyl-t~~~~~~.Similar trends were observed for the ~~UC~I-~RNA~~~and leucine ethyl ester pair by Wolfenden (88) and the g~yc~l-t~~~~'~and glycine ethyl ester pair by Jencks et al. (143). In general, the greater rate of hydrolysis for an aa-tRNA relative to an alkyl ester has been attributed to the presence of the hydroxyl group vicinal to the ester moiety in the aa-tRNA (144-146) (which is suggested to stabilize the oxyanion transition state by hydrogen bonding (745,
147, 148)) and the ribosyl endocyclic oxygen (88, 90) (which inductively withdraws electron density and hence rnakes the ribose moiety a better leaving group than the alkoxide). The vicinal hydroxyl group and endocyclic oxygen are responsible for a 3-fold (148) and 10-fold (88, 90), respectively, rate acceleration relative to analogues in which these elements are absent.
Though the ribosyl endocyclic oxygen, vicinal hydroxyl group, and aminoacyl side chain appear to play a part in determining the susceptibility of the ester in aa-tRNA to hydrolysis at physiological pH (-7.3),they are not the only factors. It appears that there is also an electron-withdrawing effect from the protonated a-amino group (1 5). For example, it has been determined by
Schuber and colleagues that valyl-t~~~~~'with a protonated NH:, hydrolyzes 90 times faster than does unprotonated V~I-~RNA~~'.and 55-fold faster than N- acetyl-valyl-~RNA~~'(whose a-NH is not protonated) (90). Such experiments have not been reported for cysteinyl-t~~~~~~;however, a similar outcome is likely considering the similarity in structure between the two aa-tRNAs.
In general, electrostatics do not appear to be significant in the rate of aa- tRNA hydrolysis (145, 149, 150). The terminal nucleobase and the polynucleotide chain do not appear to be important either (144, 151).
1.4.5 Stability of the aminoacyl ester bond in EF-Tu-GTP-bound CYS-~RNA~Y~
For comparison with the stability of the aminoacyl ester bond in uncomplexed C~S-~RNA~",the half-lives for EF-Tu-GTP-protected cysteinyl-
~RNA'" during hydroxide-catalyzed hydrolysis at pH 8.0 and different temperatures were determined. At 4 OC, 24 OC (rt), and 3I0C, the tins were determined to be > 18 hl >> 2.5 h, and -1 -5 hl respectively (table 1.3 and figure
1.20). O 5 10 15 20 O O. 5 1 15 2 2,5 Time (hour) lime (hour)
O 0.5 1 1.5 2 2,5 Time (hour)
Figure 1.20. Line graph for amount of intact [35S]-Cys-tRNACYs-EF-Tu-GTPternary complexes remaining at different time intervals during chemically hydrolysis at pH 8.0 and 4 OC (A), 24 OC (B), and 31 OC (C). Table 1.3. Half-lives for EF-Tu-GTP-protected C~S-~RNA'"at different pHs, ternperatures, and buffer compositions.
Buffer PH Temperature (OC) Half-l ife (h)
50 mM NaH2P04, 8.0 4 > 18 300 rnM NaCI, 500 mM imidazole
50 mM NaH2P04, 8.0 24 ut) 300 mM NaCI, 500 mM irnidazole
50 rnM NaH2P04, 8.0 31 1.5 300 mM NaCI, 500 mM imidazole
When these data are compared with those for factor free C~S-~RNA~~~
(table 1A), it is evident that EF-Tu-GTP-bound C~S-~RNA'~is more resistant than uncomplexed C~S-~RNA~~~towards chernical hydrolysis.
In the X-ray crystallographic structure for C~S-~RNA~"-EF-TU-GDP NP reported by Nissen and colleagues (34). the 2'-OH group participates in a hydrogen bond with the carboxyl group of Glu271 (figure 1.1 1). Because of this interaction. the 2'-hydroxyl group is no longer available for stabilization of the transition state for chernical hydrolysis. The free amino group is hydrogen bonded by the main-chain amide of His273 (donor) and the main-chain carbonyl group of Asn285 (acceptor) (figure 1.1 1). Since the amino group accepts a hydrogen bond from His273, it cannot be protonated as an alkyl-ammonium ion, as it would be in free solution. It has been proposed that the neutral state of the free amino group decreases the electrophilic character of the carbonyl carbon and therefore the susceptibility to nucleophilic attack by hydroxide (88-90). Also, by docking into the binding site of GTP-bound EF-Tu, the aminoacyl ester bond
of cysteinyl-~RNA'" is sterically protected frorn the surrounding aqueous
environment.
1A.6 Characterization of chemically-modified C~S-~RNA~~*
The cysteinyl side chain of C~S-~RNA~~~was alkylated with [I4c]-
iodoacetamide and three different fluorescent iodoacetamide derivatives (figure
1-1 6a, b, and c) using the conditions outlined in scheme 1.4A. As a control in these reactions, unaminoacylated ~RNA'~was treated with each of the iodacetamide derivatives under the same conditions.
The level of DMSO was an important factor. In a series of labelling reactions with [14~]-iodoacetamide,it was found that c 47% DMSO did not cause labelling of the tRNA whereas with 75% DMSO,reaction with the tRNA as well as the cysteinyl side-chain (data not shown) was observed.
In another experiment, it was observed that prolonged exposure of Cys-
~RNA'" to [l-14~]-iodoacetamide (overnight) at room temperature resulted in labelling of the tRNA backbone (data not shown).
In both cases, background labelling is presumably a result of reaction of the iodoactamide function with the 4-thiourdiine base (s4u) at position 8 of E. coli
~RNA'" (scheme 1.4B) (152, 153).
Using the conditions in scheme 1.4A, 70% of ~~steinyl-t~~~~~~was successfully converted to a chemically-modified C~S-~RNA'"and no significant chemical hydrolysis was detected (see section 1.3.1 0 for method of calculation). Kabat and colleagues used different conditions to label yeast C~S-~RNA~" with N-(1 -oxyl-2,2,5,5-tetramethyl-3-pymolidinyl)iodoacetamide (scheme 1-2A)
(121). They carried out the reaction at 37 OC in a 10% acetone solution, pH 7, saturated with the labelling reagent for 35 min. The high temperature and 10% acetone were employed to dissolve the nitroxide reagent Reaction completion was reached within 15 - 20 min. Under the labelling conditions, the ester Iinkage in the aa-tRNA was found to hydrolysed with a half-life of 118 min and non- specific labelling of the tRNA backbone was detected. Since the tRNA does not contain any thiolated bases nor other sfrong nucleophiles, it was presumed that the site of attachment on the tRNA was with weak nucleophiles which were present at high levels. The aforementioned labelling conditions were employed for our experiments so as to avoid labelling of the 4-thiouridine base.
The conditions used by Ohtsuka and colleagues to derivatize cys-t~~AC* with biotin-HPDP or biotin-BMCC (schemes 1.2A and 6,respectively) (122) were not described in detail and were reported after our work had already been carried out.
C~S-~RNA~~*reacted with radiolabelled iodoacetamide was characterized by liquid scintillation counting. A 1 PL aliquot from a sample containing -28 pM
['4~]-iodoacetamide-derivatizedC~S-~RNA~~~ typically gave a count of 512 (k 30) cpm while that from an aliquot containing an equivalent amount of unarninoacylated tRNA that had been treated with the labelling reagent produced a 28 (t5) cpm count. Background cpm was measured at 28 (+ 5). The fluorescently-labelled C~S-~RNA~~~constructs were identified by
fluorescence imaging on a UV transillurninator (figure 1.21). The fiuorescein-
labelled compound (figure 1.21, lane e) and the IAEDANS derivative (figure 1.21.
lane f) appeared as fluorescent bands that migrated in the expected position on a
10% 8 M urea/TBE polyacrylamide gel. The fluorescence of AIASS-labelled
cysteinyl-~RNA'~was diffwlt to observe with a standard UV transilluminator
unless a large amount of the tRNA construct was analyzed. Therefore, instead
of fluorescent imaging, AIASS-modified C~S-~RNA~~~was obsewed by
fluorescence spectroscopy using a fluorescence spectrophotometer (figure
1-22b). The previous two fluorescently-labelled C~S-~RNA~~~constructs were
identified by fluorescence spectroscopy as well (figures 1.22a and c). No
significant background fluorescence was detected when unaminoacylated
~RNA'" was treated with any of the fluorescent labelling reagents (figures 1.21,
lane g, and 1.22) using the conditions described in the experimental section (vide
ultra: section 1.3.10).
On the 10% 8 M urea/TBE polyacrylamide gel, it was also obsewed that
the fluorescence due to the IAF, AISS, and IAEDANS moieties in their respective
modified C~S-~RNA~~~constructs gradually weakened with time and disappeared
approximately 2 h after the electrophoretic separation. This was presumably a
result of chemical hydrolysis of the aminoacyl ester bond, as the pH of the gel was -8.0. The rate of fluorescence disappearance was in line with the rate of
C~S-~RNA~~'hydrolysis at pH 8.0 and 22 OC. The Ruorescence disappearance disulphide-linked Cys-tRNACYs
unacylated and arninoacylated ~RNAQs
Figure 1.21. A composite of an autoradiograph (lanes a and b) and a fluorescence image (lanes c - g) of a 10% 8 M urea/TBE polyacrylamide gel. Lane a: [35S]-Cys-tRNACYSbefore treatment with TCE P; lane b: after treatment with TCEP; lane c: (IAF)-labelled Cys-tRNACYSstained with ethidium bromide; lane d: unaminoacylated tRNACYsstained with ethidium bromide; lane e: (IAF)-treated Cys-tRNACYs(unstained); lane f: (1AEDANS)-treated Cys-tRNACYS(unstained); and lane g: unaminoacylated tRNACYStreated with IAF (unstained). Figure 1.22. Fluorescence emission scans of labelled tRNAs (solid Une) together with unlabelled controls (broken line) (unaminoacylated tRNACYstreated with the labelling reagents under the same conditions as in experiments with Cys-tRNACYs)(approx. 0.5 pM each). (a) IAF- labelled Cys-tRNACYSin a buffer containing 50 mM Tris, pH 7.5 (HCI), 30 mM KCI, and 10 mM MgCI, (excitation at 492 nm); (b) AIASS-labelled Cys-tRNACYSin a buffer made up of 10 mM NaOCOCH,, pH 4.5 (CHJOOH) (excitation at 329 nm); and (c) IAEDANS-labelled Cys-tRNACYSin a buffer consisting of 10 mM NaOCOCH,, pH 4.5 (CH,COOH) (excitation at 336 nm). provides further evidence that the fluorophores were attached to C~S-~RNA~~via
the cysteinyl side chain.
1.4.7 Interaction of [35~]-~ys-t~~~Cysand chemically-modified C~S-~RNA~~'with EF-Tu(NHis6)-GTP and EF-Tu(NHis6)- GDP
Each of the chemically-modified C~S-~RNA~"constructs were tested for
interaction with EF-Tu, tagged with an N-terminal hexa-histidine group, both in the GDP-bound and GTP-bound states. The EF-Tu protein used in these binding experiments was of T. fhermopilus origin. The prirnary sequence of E. coli elongation factor Tu shares extensive homology with EF-Tu from T. thermophilus and T. aquaticus (38, 154, 155). The sequences of the elongation factor from the latter two species differ in only 10 of 405 residues (1 55) and the proteins from these species have the advantage of being temperature stable. The crystal structure reported by Nissen et al. (34) for the ternary complex of C~S-~RNA~~-
EF-Tu-GDPNP employed EF-Tu from T. aquaticus. We employed EF-Tu from T. thermophilus as this was available with a nickel affinity tag and would permit cornparison of experimental binding data with the reported structure.
We examined the ability of EF-Tu-GTP to form ternary complexes with
[14~]-iodoacetamide-derivatizedC~S-~RNA~" and the fluorescently-labelled Cys-
~RNA'~constructs using a simple binding assay. The modified tRNAs were cornbined with EF-Tu-GTP in solution, which was then mixed with a nickel affinity matrix. In this manner, EF-Tu-GTP molecules, along with aminoacylated tRNA constructs that interact with them, became imrnobilized on the nickel resin. lmmobilized EF-Tu-GDP, which was expected to bind [35~]-~ys-t~~~C~weakly, was used as a control(l09) in these experiments- After washing the resin, the protein was eluted with a buffer containing concentrated imidazole. The presence or absence of fluorescently-labelled tRNA associated with EF-Tu was then assayed by fluorescemce spectroscopy at waveIengths appropriate for the individual fluorophores; and the presence or absence of radiolabelled acetamido-
C~S-~RNA'" associated with EF-TU was assayed by liquid scintillation counting.
Elution profiles were obtained in al1 cases (figures 1.23 - 1.26).
For al1 four modified tRNAs, radioactivity due to the acetamido group and fluorescence due to the flulorescent tags were only observed in the ternary complex when EF-TU was in the GTP form (figures 1.23 - 1.26 (red curves)). As expected, unmodified [35~]-~ys-t~~~Cpwas also only associated with the EF-
Tu-GTP cornplex (figure 1.27B (red)). No interaction was observed with any of the tRNAs and EF-Tu-GDP (figures 1.23 - 1.27B (blue curves)). Instead, in the
GDP case, al1 the radioacti vity or fluorescence was observed in the flow-through frorn the affinity colurnn. A lack of interaction in the GDP case was not due to deacylation of the tRNAs since 73% of the flow-through was recovered as intact aminoacylated tRNA for [35~]-~ys-t~~~C",as determined by ethanol precipitation and liquid scintillation counting cf the flow-through (data not shown).
The remaining 27% radioactive material was characterized as free cysteine that had arisen presumably frorn deacylation of ~~stein~1-t~~~~~~.The extent of deacylation detected here ris consistent with the measured deacylation rates for uncomplexed C~S-~RNA~"aï pH 8.3 and 4 OC (table 1-1 and figure 1 -19A). The same results were observed for 6 independent EF-Tu-GDP binding experiments I I Concentrated ,
O 5 10 15 20 25 30 35 fraction
Figure 1.23. Elution profiles from GDP- (blue) and GTP-bound (red) EF-Tu binding assays for [l-14C]-acetamido-Cys-1RNACVS. The concentrations of EF-Tu and tRNA construct in the 1 mL bindnig reaction were -1 1 pM and -1.5 PM, respectively. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution. Concentrated v0.- imidazole OH
O 5 10 15 20 25 30 35 Fraction
Figure 1.24. Elution profiles from GDP- (blue) and GTP-bound (red) EF-Tu binding assays for IAF-C~S-~RNA~Y~.The concentrations of EF-Tu and (RNA construct in the 1 mL binding reaction were -21 PM and -1.5 PM, respectively. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution.
r 30000 =L 8 2sooo T Concentrated 2 20000 imidazo le a Ci i A .-z 15000 E g 10000 rn C r 5000 3 S O O 5 ! 10 15 20 25 30 35 Fraction
Concentrated imidazole
O 5 IO 15 20 25 30 35 Fraction
Figure 1.27. Elution profiles from E. coli (A) and T. thermophilus (B) GDP- (blue) and GTP-bound (red) EF-TU binding assays for [35S]-Cys-tRNACyS.The concentrations of EF-TU and tRNA construct in the 1 mL binding reaction were -1 1 FM and -2 PM, respectively, for the experiment with E. coli EF-TU; and -21 pM and -2 FM,respectively, for the experiment with T. thermophilus EF-TU. Fractions 1 - 21 were collected during column washes while fractions 22 - 31 were collected during elution. with [JS~]-cysteinyl-t~~~C?Overall, the binding experiments indicated that acetamido-labelled C~S-~RNA'~and ail three fluorescently-labelled tRNAs interact specifically and effective1y with EF-TU-GTP.
Radioactivity and fluorescence were observed in the flow-through for the
GTP experiments as well, albeit to lesser degree than that from the GDP experiments (approx 50% of total radioactivity or fluorescence detected in the flow-through) (figures 1-23 - 1-278 (red curves)). For [35~]-~ys-t~~~CF,37% of the flow-through was from intact aminoacylated tRNA that had failed to bind to
EF-TU-GTP on the nickel agarose resin, while 63% was from free cysteine that had become detached from ~RNA'" (as detenined by ethanol precipitation and
Iiquid scintillation counting). This type of elution profile for the binding between
[35~]-~ys-t~~~Cpand EF-Tu(NHis6)-GTP (GTP-bound EF-Tu(NHis6)) waç observed in 9 independent experirnents.
The occurrence of intact aminoacylated tRNA in the flow-through for the binding experiments with GTP-bound elongation factor Tu and each of the chemically-modified C~S-~RNA~"constructs was not due to chemical modification of the cysteinyl side-chain, as the levels detected were similar to those from assays with unmodified [35~]-~ys-t~~~CF.
The EF-TU-GTP and EF-Tu-GDP binding experiments for [35~]-~ys-
~RNA~"were then repeated with 10-fold less [35~]-~ys-t~~~CySand the same arnount of elongation factor (figure 1.28). Elution profiles from these experiments are similar to the originals (figure 1.278). indicating that column saturation was not the cause for incomplete binding of the aminoacylated tRNA. Concentrated imidazole
Figure 1.28. Elution profiles frorn GTP-bound EF-Tu binding assay for [35S]-Cys-tRNACys.The concentrations of EF-Tu and tRNA construct In the 1 mL binding reaction were -21 VM and -0.2 FM, respectively. Fractions 1 - 21 were collected during colurnn washes, white fractions 22 - 31 were collected during elution. In addition, no EF-TU protein could be detected in the fiow-throughs, as judged by SDS-PAGE. The result also indicates that inefficient binding of ternary complexes to the nickel affinity matrix was not the reason for the incomplete trapping of aminoacylated tRNA molecules on the Ni-NTA agarose column.
It has been reported by Ribeiro and colleagues (105) that T. thermophilus
EF-Tu tagged with an N-terminal hexa-histidine tag is "less efficienf' than its C- terminal his-tagged counterpart towards binding to L~S-~RNA~".
We also examined binding of [35~]-~ys-t~~~CFto E. col; EF-TU tagged with a C-terminal hexa-histidine moiety (figure 1-27A). The radioactivity detected in the flow-through relative to that from the ternary complexes in the GTP experiment (49%) was much less than that observed in the experiment with EF-
Tu(NHis6)-GTP (-50%). The elution profile from the GDP experiment is the same as the original. These results are consistent with the report by Ribeiro and
CO-workersand suggests that N-terminal modification of EF-Tu decreases the ability of the elongation factor to form a stable ternary complex with GTP and
C~S-~RNA~~~.It has been shown that EF-TU-GTP bearing a his-tag on its C- terminus has a dissociation constant which is the same as that of the unmodified
EF-Tu towards L~S-~RNA~~(105). Perhaps N-terminal modification induces conformational changes in the EF-TU protein tertiary structure which negatively affects the ability of EF-Tu to interact with unmodified and chemically-modified
C~S-~RNA~?In the 3-0 structure (34),the N-terminus is near the aminoacyl- tRNA binding site whereas the C-terminai is far removed. In the experiments described up to this point, formation of ternary complexes between GTP-bound EF-Tu molecules, with either a C-terminal or an
N-terminal his-tag, and arninoacylated tRNA constructs were carried out in solution. The ternary complexes were subsequently immobilized on the nickel affinity column for separation. Since placement of an hexa-histidine tag at the N- terminus of the elongation factor was found to affect the strength of aa-tRNA association, it was plausible that irnmobilization of the T. thermophiius EF-
Tu(NHis6) protein on a nickel affinity matrix prior to binding of aminoacylated tRNA, as opposed to after, could have an effect on the extent of ternary cornplex formation.
To investigate this matter, binding experiments with GTP-bound EF-
Tu(NHis6) and the fluorescently-labelled C~S-~RNA~~~constructs were repeated using an assay in which modified tRNA was combined with EF-TU-GTP that had been pre-immobilized on a nickel affinity resin. Fluorescence scans for the eluted ternary complexes are shown in figures 1-29a - c (solid line). Experiments with the GDP-bound form of EF-Tu were performed as well. For comparison purposes, [3s~]-~ys-t~~~CFwas treated in the same manner and the presence of [35~]-~ys-t~~~C~~~-~u-~~~was detected by liquid scintillation counting
(figure 1-29d).
For al1 three modified tRNAs, fluorescence due to the fluorescent tag was only observed in the ternary complex when EF-Tu was in the GTP form. The fluorescein label gave the strongest signal primarily because the molar extinction coefficient of fluorescein is the highest of the three (emax(IAF) = 75 000, Emax Wavelength (nrn) Wavelength (nrn)
~50052aW560 Wavelength (nm) Figure. 1-29. Detection of ternary complexes. Fluorescence ernissbn scans (excitation as in figure 1.22) of Cys-tRNACYSderivatives associated with EF-Tu-GTP (solid line) (a = IAF-Cys-tRNACYS,b = AIASS-Cys-tRNACYs,and c = IAEDANS-C~S-~RNA~Y~)eluted from Ni-NTA columns as described in experimental procedures (section
1.3.1 1.4). EF-Tu-GDP control is shown in each case (broken line), . measured under identical conditions. Some residual fluorescence from the elution buffer is observed between 450 and 550 nm in al1 cases. All scans are averages of three independent binding experiments. Panel ci shows counts per minute associated with [35S]-cysteinein the EF-Tu-GTP (solid) and EF-TU-GDP (open) samples. (AIASS) = 39 000, E,, (IAEDANS) = 5700) (figures 1-29a - c (solid line)) (118).
A small blue shift in the maximum emission wavelength of the IAEDANS
derivative (2 nm) presumably reflects the effect ofthe EF-Tu binding pocket on
this environmentally sensitive fluorophore (1 18). As expected, unmodified f3%]-
C~S-~RNA~~was only associated with the GTP complex (figure 1.29d (solid
bar)). No interaction was observed with any of the tRNAs and EF-Tu-GDP
(figures 1.29a - c (broken line), and d (open bar)I. Instead, in the GDP case, al1
the fluorescence or radioactivity was observed in the flow-through from the
affinity column. A lack of interaction in the GDP case was not due to deacylation
of the tRNAs since intact aminoacylated tRNA was recovered in the flow-through
(data not shown). Flow-through was also obserwed for the GTP experiments.
albeit with an amount that was much less than that found from the GDP
experiments, at a proportion that was similar to that found in the EF-Tu-GTP
binding experiments using the other binding assay (vide ultra). These data confirm that al1 three fluorescently-labelled tRNAs interact effectively with EF-Tu-
GTP.
Since the results from the latter binding experiments (ternary complex formation with nickel agarose pre-imrnobilized EF-Tu-GTP) are similar to those from the binding assays in which ternary complex formation was carried out in solution (vide ultra), immobilization of the his-tagoged EF-Tu-GTP on the nickel affhity rnatrix had no observable effect on the association of aminoacylated tRNA with the elongation factor. While the present assays cannot furnish quantitative data on association
constants of the complexes, it is likely that only a severely impaired interaction
between a chemically-modified aminoacyl-tRNA and EF-TU would prevent
incorporation of the amino acid into protein. After all, variations in association
constants by more than an order of magnitude are observed among the naturally-
occurring aminoacyl-tRNAs (25).For practical purposes, a minor change in
interaction affinity could probably be cornpensated simply by adding more of the
chemicalty-modified aa-tRNA to the protein synthesis reaction.
1.4.8 The nature of the amino acid binding site on EF-TU
The finding that al1 three fluorescently-modified C~S-~RNA~~~species bind
to EF-Tu irnplies the arnino acid binding site of EF-Tu witl tolerate considerably
greater diversity in side-chain structure than that which occurs with the naturally-
occurring amino acids. We therefore decided to model the structure of the
bulkiest of these constructs - fluorescein-labelled C~S-~RNA'"- in an effort to
understand how this occurs.
Figure 1.30 shows surface representations of the amino acid binding
pockets observed in the two crystallographically-determined ternary complexes of EF-Tu-GDPNP. ~he-~RNA'"-EF-TU-GDPNP (figure 1.30A) (33) and Cys-
~RNA~~'-EF-TU-GDPNP(figure 1.308) (34). The EF-TU protein surface is shown together with stick representations of the terminal adenosine and amino acid attached at the 3' position. EF-TU residues making contacts with the aminoacyl group are labelled. Some differences are observed between the two ternary complexes, in particular. the side-chain of Arg274 moves significantly. This Figure 1.30. Structures of the amino acid binding pocket of EF-Tu complexed with Phe-tRNAPhe(A) and fluorescein-labelled Cys-tRNACyS(B). The protein is shown in surface representation and the terminal adenosine group with the amino acid esterified at the 3' position is shown in stick representation. Atom colours are blue (nitrogen), white (carbon), red (oxygen), and yellow (sulphur). Hydrogens are truncated for clarity. The chemical structure of the fluorescein derivative is shown in figure 1.16a. The rest of the tRNA molecule extends to the right from the terminal adenosine group but is removed for clarity. movement together with movements of the side-chains of tiis67, Asn285, and
Glu226 help to maximize contacts between the side-chain of the aminoacyl group
and the protein. Interactions involving the backbone of the aminoacyl group are
more conserved. In particular, the a-amino group makes interactions with the
backbone NH of His273 and the backbone carbonyl oxygen of Asn285 (34,40).
As described in the experimental section, a fluorescein group was
modelled into the binding pocket to explore the volume accessible to chernical
modifications ai the cysteine side-chain, The structure of the adduct was energy minimized allowing only the chemically-modified side chain to move. This is a rather restrictive requirement as protein side-chains (e-g. Arg274) are clearly able to move to accommodate different structures in the amino acid binding pocket
(compare figures 1.30A and 1.30B). The minimization routine quickly finds a conformation in which no serious steric clashes or unfavourable electrostatic interactions occur. This does not imply that the conformation depicted is the most stable, only that such a conformation is energetically reasonable.
Inspection of the structure reveals that, if the chemically-modified side-chain is narrow and flexible enough to escape the immediate vicinity of the 3' attachment site, Iittle hindrance is presented by EF-TU as the binding pocket widens rapidly out into bulk solution. Since al1 the chemica! modifications we tested are relatively narrow structures at the cysteine attachment site, the structure shown in figure 1.308 provides a straightforward explanation of the observation that al1 three chemically-modified cysteine derivatives bind to EF-TU. In addition, the structure rationalizes the ease of incorporation of biotinylated and fluorescently- labelled lysine derivatives into proteins (128, 156).
One rnight extend the predictions based on the model shown in figure
1.308 to suggest that non-natural amino acids that would be expected to have the most diffrculty in associating with EF-Tu would be ones in which there were bulky groups or unusual H-bond donorlacceptor sites very close to the amino acid backbone. For instance, it is immediaiely evident that there is limited space for a D-amino acid side chain to bind in the pocket without a rotation that would alter the conserved interactions of the a-amino group. Thus, the substantially decreased affinity of D-~~~OS~I-~RNA~~(29, 96), and the inability of D- phenylalanine (Phe) to be incorporated into proteins (15, 157) are readily explained. Diminished interactions with EF-Tu may also explain the failure of 1- pyrenylalanine to be incorporated into proteins (10). The structure of the binding pocket does not, however, explain the report of Cornish et al, that a dansylated arnino acid with a structure similar io IAEDANS derivative studied here failed to incorporate (5). 1.5 References
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Reverse-Phase High Performance Liquid Chromatography of PartiaIIy-Purified ChemicaIIy-Modified Cys-tRNACYS 2.1 Introduction
In chapter 1, it was shown that when E. colit~~~'~was aminoacylated
with cysteine using the cognate cysteinyl-tRNA synthetase and ATP as a
cofactor, approximately 40% of the tRNA became charged. For synthesis of a
chernically-modified C~S-~RNA'" construct ([I-14~]-acetamido-~ys-t~~~Cys, IAF-
C~S-~RNA~",AIASS-C~S-~RNA~~, or IAEDANS-C~S-~RNA~~~ (chapter 1, figure
1-16}),C~S-~RNA~~, along with the unseparated unaminoaccylated tRNA, was
subjected to a reaction with an iodoacetamide derivative. The reaction was
carried out under mild conditions such that just the cysteinyl side chain of the
aminoacylated tRNA was labelled with the labelling reagent. Because of the mild
labelling conditions, only 70% of the charged tRNA was converted to chemically-
modified cysteinyl-~RNA'? A crude sample of chemically-modified C~S-~RNA'" was thus cornposed of approximately 60% unacylated ~RNA'", 28% chemically-
modified C~S-~RNA~~~,6% unlabelled C~S-~RNA~~~,and 6% disulphide-linked
C~S-~RNA~".Without an additional purification step to remove unacylated
~RNA'", unlabelled C~S-~RNA'F,and disulphide-linked C~S-~RNA~~~,the modified tRNA was subjected to binding experiments with GDP-bound and GTP- bound elongation factor Tu. Fractionation of the different tRNA constructs so as to isolate a single species of the modified tRNA prior to performing the binding assays was not necessary as the other tRNA constructs did not have an observable effect on the interaction of chemically-modified cys-t~~ACFwith EF-
Tu-GTP and EF-Tu-GDP. However, it would be beneficial if each of the different components of a crude labelled C~S-~RNA~~sample could be fractionated since separation of the different ~RNA'~~species would make possible regeneration of
unaminoacylated ~RNA'~for multiple rounds of derivatization. In addition,
purification of a chemically-modified cys-tR~ACrjis mandatory for ~RNA'~~
mediated labelling of proteins with non-natural reporter groups (discussed in
chapter 3).
It has been shown by several investigators that I~S~I-~RNA~~~(Lys-
~RNA~~~)chemically modified with pazidobenzoic acid (ABA), 5-azido-2-
nitrobenzoic acid (ANB), 4-(3-trifluoromethy1diazirino)benzoic acid (TDBA), biotin,
N-biotinyl-6-aminohexanoic acid (AhxBio), or 6-(7-nitrobenz-2-oxa-1,3-diazoI-4-
y1)aminohexanoic acid (NBDAhx) (figures 2.1A, B, C, Dl El and F, respectively)
can be separated from unaminoacylated ~RNA~~and unmodified L~S-~RNA~~~by
benzoylated diethylaminoethyl (DEAE) cellulose (BD cellulose) column
chromatography (1-5).
In experiments conducted by Kabat and colleagues, ~~stein~l-t~~~~~
labelled with the nitroxide spin labeling reagent N-(1 -oxyl-2,2,5,5-tetramethyl-3-
pyrrolidinyl)iodoacetamide(chapter 1: scheme 1.2A) was enriched 6-fold after
purification by chromatography on a BD cellulose colurnn (6).
BD cellulose is a derivative of DEAE cellulose in which the hydroxyl groups are benzoylated. The modification increases hydrophobie interactions between the resin and polynucIeotides, at the sarne time preserving the physical properties necessary for column chromatography (7).
Reverse-phase high performance liquid chromatography (RP-HPLC) on a
C3 column has been used succeçsf~llyby Odom and colleagues (8) for the fractionation of different tRNA constructs in a sample containing coumarin- labelled [35~]-methionyl-t~~~N"(M~~-~RNA~~') (CPM-SAC-M~~-~RNA~~~, where
CPM = 7'diethylamino-3-(4'-maleimidylphenyl)-4hycoumarin)) (figure 2.2A), deacylated ~RNA~~',unreacted mer~a~toacet~l-~~~]-~et-t~N~~(SAc-Met-
~RNA~~~)(figure 2.2B), disulphide-linked rnercapt~acetyl-[~~~]-~et-t~N~~~~
(figure 2-26},and non-specifically labelled ~RNA~.RP-HPLC has also been utilized to puriv methionyl-t~~~~~'labelled with BODIPY-FL (4,4-difluoro-5,7- dimethyl-4-bora-3a14a-diaza-s-indacene propionic acid) (figure 2.2D) (9).
In this chapter, we describe reverse phase high performance liquid chromatography on a C8 column of a crude sample of each of the fluorescently labelled C~S-~RNA~"constructs used in the binding experiments in chapter 1
(IAF-labelled C~S-~RNA~",AIASS-labeiled C~S-~RNA~~~,and IAEDANS-labelled
C~S-~RNA~~),as well as a biotin-labelled C~S-~RNA~~,and describe how the desired tRNA species can be purified successfully. Figure 2.2. Structural formulae of the 3' end of C PM-SAC-Met-tRNAWet(A), mercaptoacetyl-M~~-~RNA~(B), disulphide-linked rnercaptoacetyl- M~~-~RNA~='(C), and BOD I PY-FL-labelled methionyl-tRNAmet(D). 2.2 Materials
B IADD ((+)-biotinyl-iodoacetamidyl-3,64ioxaoctanediamine) (an iodoacetamide derivative of biotin), lrnmuno~ure"alkaline phosphatase- conjugated streptavidin, and an immuno~ure@Fast Red TR/AS-MX alkaline phosphatase conjugate substrate kit were purchased from Pierce (Rockford, IL).
An NBTIBCIP (Le- nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) alkaline phosphatase conjugate substrate kit was purchased from BIO-RAD
(Mississauga, ON). HPLC grade (Optima) methanol was purchased from Fisher
Scientific Limited (Nepean, ON). Biotinylated 1ysyl-t~~~~~~(~ranscend~~tRNA) was purchased from Promega (Madison, Wi). AI1 other chemicals were obtained as described in chapter 1 (section 1.2). 2.3 Experimental Procedures
2.3.1 Synthesis of IAF-labelled, AIASS-labelled, IAEDANS- labelled, and BIADD-labelled cysteinyl-t~~~~~'
The cysteinyl side chain of ~-c~stein~l-t~~~~~~was alkylated with four
different thiol-reactive cornpounds: IAF (chapter 1: figure 1.16A); AIASÇ
(chapter 1: figure 1-1 6B); IAEDANS (chapter 1: figure 1-1 6C). and (+)-biotinyl-
iodoacetam idyl-3,6-dioxaoctanediamine (ez-linkTMpeo-iodoacetyl biotin) (B IADD)
(chapter 1: figure 1.16D). The modified tRNA constructs were synthesized and
partially purifed by a series of ethanol precipitations in the same manner as
described in chapter 1 (section 1.3.5: "Aminoacylation of ~RNA'~'with L-
cysteine"; and section I-3.10: "Chemical modification of C~S-~RNA~Y~with [l-
'4~]-iodoacetarnide,IAF. AIASS, and IAEDANS"). For the chernical modification
reaction with BIADD, the concentration of labelling reagent in the 100 pL reaction
volume was -37 mM, while C~S-~RNA~~~was present at the usual 20 pM level.
Labelling reactions in which ~~stein~l-t~~~~~~was replaced with unacylated
~RNA'Y', which served as control experirnents, were performed in parallel with the alkylations of C~S-~RNA~~~.The purpose for the control reactions was to check for non-specific labelling of the tRNA itself. Each crude chemically- modified C~S-~RNA~~~construct was dissolved and stored in a buffer containing
10 mM NaOCOCH3, pH 4.5, at a concentration of approximately 28 PM. By
calculating the yield of C~S-~RNA~~~from the aminoacylation reaction (chapter 1:
section 1.3.5) and then the yield of labelled C~S-~RNA~~~in a reaction with [l-
'4~]jodoadematide(chapter 1: section 1.3.1 O), it was estimated that each of the aforernentioned labelled tRNA sarnples was composed of -60% unacylated
~RNA~~',-28% chemically-modified C~S-~RNA~~~,-6% unlabelled C~S-~RNA~~~. and -6% disulphide-linked C~S-~RNA~~~.
A 3 pL aliquot of C~S-~RNA~~'with a biotin tag and the same amount of unaminoacylated ~RNA~~'treated with the biotin iodoacetamide were characterized by Northern blot (vide infra: section 2.3.2). Al1 other labelled tRNAs were characterized by fluorescence imaging in the same manner as described in chapter 1 (section 1.3.10).
The crude modified tRNAs were either used directly in RP-HPLC experimentç or stored in a -80 OCfreezer.
2.3.2 Characterization of BIADD-labelled C~S-~RNA~~~by Northern blot
Crude BIADD-C~S-~RNA~~~(BIADD-labelled C~S-~RNA~~~)was separated on a 10% 8 M urea/TBE polyacrylamide gel in the same manner as described in chapter 1 (section 1.3.6). After electrophoresis. the gel was rinsed in -15 mL of
1 X TBE for -15 min on an automated rotatory shaker at room temperature.
Bands on the gel were transferred to a piece of nylon membrane with fixed positive charges (Zeta-Probe GT@)(BIO-RAD) by transfer blotting using a Mini
~rans-Hot@Electrophoretic Transfer Cell (BIO-RAD). The transfer sandwich and electrophoretic system were assembled according to the manufacturer's instructions. The transfer was carried out in 1 X TBE at a constant voltage and current of 90 V and 350 mA, respectively. The duration was 1 h and the temperature was 4 OC. A stirring bar was included in the transfer assembly to allow for equal distribution of heat during transfer. After blotting, the membrane was separated from the transfer assembly and placed in -1 5 mL of a solution containing 20 mM Tris-CI, pH 7.5, and 150 mM NaCl (TBS (Tris-buffered saline)) in a shallow dish, which was placed on a rotatory shaker at room temperature for
-5 min. Five minutes later, the TBS was discarded and -15 mL of a solution consisting of TBS and 7% SDS (TBSS) was added. The membrane was rinsed in this solution on the shaker for -1 h. The purpose of this step was to use SDS rnolecules to block al1 unoccupied sites on the membrane. In this way, there should be less background from the colorimetric assay to be performed later
(vide infra). After 1 h, the TBSS was discarded. To the membrane was added a fresh -15 mL aliquot of TBSS with afkaline phosphatase-conjugated streptavidin
(3 pg/mL). The membrane was rinsed with the TBSSfstreptavidin-alkaline phosphatase solution for -1 h. In this step, the alkaline-phosphatase conjugated streptavidin molecules should bind to any membrane-bound molecules containing an exposed biotin tag. One hour later, the TBSSlstreptavidin-alkaline phosphatase solution was discarded. The membrane was rinsed twice with two separate aliquots of -15 mlof TBSS (-1 min each) and twice with two separate aliquots of -15 mL of TBS (-1 min each). Ten millilitre of a solution containing alkaline phosphatase conjugate substrate (NBT/BCIP (scheme 2.1A) or lmmuno~ure@Fast Red TR/AS-MX (scheme 2.1 B)), prepared according to the manufacturer's instructions, was added to the membrane. In the presence of the
NBTIBCIP substrate solution, bands with a biotin tag appeared purple; pink bands were detected when the other substrate (Fast Red TRIAS-MX) was 0 4' d~~q~~~ CH^ naphthol AS-MX phosphate ? BClP (5.brom.4~chloro~3.lndolyl phosphate)
alkaline phosphatase
alkaline phosphatase
NBT (nitroblue letrazoliurnchlorlde) Fast Red TR salt ci
1 resultlng preclpitatlng azo dye
Scheme 2.1. Reaction between naphthol AS-MX phosphate and Fast Red TR salt (A), and between NBT and BClP (B), as catalyzed by alkaline phosphatase. employed. The membrane was rinsed in the substrate solution until the bands were visible (-15 min). After bands were detected at a reasonable intensity, the substrate solution was discarded while the membrane was rinsed twice with two separate aliquots of -1 5 mL of H20(-5 min rinse each). The membrane was air- dried (overnight), taped onto a piece of whatrnana filter paper, and then stored in an manila envelop. Protection of the blot from light was important because light exposure caused discoloration of the bands.
2.3.2 RP-HPLC conditions for fractionation of crude chernically-modified Cys-tRNACYS
Reverse phase high performance liqüid chromatography experiments were perforrned using a Perkin Elmer HPLC system that was equipped with
Diode Array Detector 235C, a series 200 purnp, and controlled with Turbochrom
Navigator software. Separation was perforrned on a ZORBAX@RX-C8 (25 cm X
4.6 mm) column (Hewlett Packard).
Mobile phases were prepared as follows: Solution A was composed of
100% HPLC grade methanol. Solution B consisted of 20 mM NaOCOCH3, 10 mM Mg(OCOCH3)2,and 400 mM NaCI, buffered at pH 4.5 with CH3COOH. The acidic pH was necessary to minimize chernical hydrolysis of the aminoacyl ester bond in C~S-~RNA~~'and chernically-rnodified C~S-~RNA~~'.Both solutions were assembled using deionized HzO (Millipore) and pasçed through 0.22 pm filters in presterilized vacuum driven disposable filtration systems (Millipore) before use.
Solution G was Millipore deionized H20. The solutions were stored at room temperature if not in use. Unless otherwise indicated, al1 operations were performed at ambient temperatures (22 - 24 OC) and at a constant flow rate of 1 mumin.
Prior to sarnple injection, the RX-C8 column was washed in the manner
outlined in table 2-1.
Table 2.1. HPLC solvent and gradient conditions for preparation of RX-C8 column prior to sample injection.
Tme (min) %A %B %C C urve
A crude ~ampleof chemically-modified C~S-~RNA~~~was injected using a
Hamilton (Hamilton Co.) syringe that had been rinsed severai times with deionized HZO. The sample was injected into a 200 pL sample bop. If the sample volume was more than 200 pL, multiple rounds of separation were carried out. The sarnple was eluted with solvent and gradient conditions most appropriate for the rnodified tRNA (vide infra: sections 2.3.3.1 - 2.3.3.3). For each crude chemically-modified C~S-~RNA~~~construct, several d ifferent solvent and gradient conditions were tested until a satisfactory separation was achieved. 2.3.3.1 Fractionation of crude IAF-labelled C~S-~RNA~~~and charaterization of the peaks in the chromatogram
IAF-labelled C~S-~RNA'"was chromatographed using the solvent and gradient conditions specified in table 2.2.
Table 2-2. HPLC solvent and gradient conditions for fractionation of crude IAF-labelled CYS-tRNA~Y*.
Time (min) %A %B Cuwe O 1O 90 O 5 20 80 1 5 20 80 O 10 30 70 1 16 30 70 O. 10 IO0 O 1 4 100 O O 10 10 90 1
Crude IAF-labelled C~S-~RNA~~(400 pL at -28 PM) was analyzed.
Eluates corresponding to the major peaks in the chromatogram were collected as
1 mL fractions in 1.5 mL microcentrifuge tubes, which were kept on ice to minimize chemical hydrolysis of the aminoacyl ester bonds. After the elution was complete. the tubes were immediately placed in a SC1 10 speed vac (Savant) equipped with an RVT 100 refrigerated vapor trap (Savant) for 45 min to evaporate the methanol. The fractions were frozen on dry ice and then transferred to a %O OCfreezer for overnight storage. The next morning, the fractions were thawed on ice (-10 min). All fractions corresponding to the same peak in the chromatogram were pooled. After pooling, each sample was transferred to a 4 or 15 mL (depending on the volume required) ultrafree centrifuga1 filtration device equipped with a 5000 MWCO porous membrane which was prechilled on ice. The volume of solution in each filter was increased to the maximum capacity of the fiiter by adding a bufTer containing 10 mM
NaOCOCH3 and 2 mM Mg(OCOCH3)2,buffered at pH 4.5 with CHjCOOH (buffer
A). The filters were centrifuged for 30 min at a speed of 3000 rpm and 4 OC in a refrigerated centrifuge. After the centrifugation, the filtrates were discarded and the filters were filled to their maximum capacity again with buffer A. The filters were centrifuged in the same manner as just described. After removing the filtrates, the retained tRNA samples were washed with buffer A in the same manner as just described one last time. After the second wash, the retained tRNA samples were transferred to 1.5 mL microcentrifuge tubes, which were kept on ice. The volume in each tube was adjusted to 270 pL with buffer A. To each tube was added 30 pL of a solution containing 3 M NaOCOCH3, pH 5.2
(CH3COOH), and 900 lof ethanol to precipitate al1 tRNA species. The tubes were pfaced in a -20 OC freezer for 30 min and then centrifuged at 4 OC and 13
000 rpm for 15 min in a microcentrifuge. Supernatants were discarded and pellets were washed with 700 pL of a solution of 70% ethanol/30% water. After air-drying for 2 min, each pellet was dissolved in 1O - 50 pL of buffer A. The presence of tRNA in each tube was detected by urea-PAGE on a 10% 8M ureafrBE polyacrylarnide gel with ethidiurn bromide staining (chapter 1, section
1.3.6). Fluorescence from the IAF moiety of IAF-labelled C~S-~RNA~~~in each tube was detected by fluorescence imaging (chapter 1, section 1.3.8).
As controls, 25 pL of unaminoacylated ~RNA'" (-1 00 pM) and 10 pL of a crude sample containing unarninoacylated ~RNA'~(-60%), unlabelled Cys- ~RNA'~'(-36%). and disulphide-linked C~S-~RNA~~~(-4%) (-40 pM total tRNA)
were separately fractionated on the RX-C8 column using the same solvent and
gradient conditions just described for the fractionation of crude IAF-labelled Cys-
~RNA'~'. Fractions corresponding to the major peaks of the chromatogram were
collected and assayed in the same manner as just described for the labelled
. tRNA. After the final ethanol precipitation, each pellet was reconstituted in IO pL
of buffer A. The presence of tRNA in each tube was monitored by urea-PAGE
with a 10% 8M urea/TBE polyacrylamide gel in conjunction with etbidium
bromide staining.
2.3.2.1 Fractionation of crude BIADD-labelled C~S-~RNA~~*and characterization of the peaks in the chrornatogram
Crude BIADD-labelled C~S-~RNA~~'was fractionated employing the
solvent and gradient conditions outlined in table 2.3.
iable 2.3. HPLC solvent and gradient conditions for fractionation of crude B IADD-labelled C~S-~RNA~?
Time (min) %A %B Cuwe O 10 90 O 5 20 80 1 15 20 80 O 10 20.7 79.3 1 5 20.7 79.3 O 15 100 O 1 5 1 O0 O O IO 1 O 90 1
A crude sample (750 pL) containing -28 pM BIADD-labelled cys-t~~ACF
was fractionated. Fractions from the major peaks in the chromatogram were collected and assayed in the same manner as described for the fractionation of
crude IAF-labelled C~S-~RNA~"(vide ultra: section 2.3.3.1 ). After the final
ethanol precipitation, each pellet was reconstituted in 20 pL of buffer A Transfer
RNA was detected by urea-PAGE on a 10% 8 M urea/TBE polyacrylamide gel
with ethidium bromide staining. Biotin from BIADD-labelled ~~stein~l-t~~~~~in
each sample was monitored by Northern blot (vide ultra: section 2.3.2).
As controls, 10 pL of unacyiated ~RNA~~'(-1 00 PM) and 10 pL of a crude
sample containing -36% unlabelled C~S-~RNA~~~,-4% disulphide-linked Cys-
~RNA'~', and -60% unaminoacylated ~RNA~~'(- 40 pM total tRNA) were
separately fractionated on the RX-C8 column using the solvent and gradient
conditions specified in table 2.3. Fractions corresponding the major peaks in the
chromatograms were collected and assayed in the same manner as described for the fractionation of IAF-derivatized C~S-~RNA~?After the final ethanol
precipitation, each pellet was reconstituted in 10 pl of buffer A. Nucleic acid was
detected by urea-PAGE on a 10% 8 M urea/TBE polyacrylamide gel in
combination with ethidium bromide staining.
2.3.3.3 Fractionation of crude AIASS-labelled and IAEDANS-labelled C~S-~RNA~~~and characterization of the peaks in the chromatograms
AIASS-labelled and IAEDANS-labelled C~S-~RNA~~were chrornatographed using the solvent and gradient conditions specified in table 2.4. Table 2.4. HPLC solvent and gradient conditions for fractionation of crude AIASS- labelled and IAEDANS-labelled C~S-~RNA~".
Time (min) %A %B Cu~e O 10 90 O 5 20 80 1 20 20 80 O 15 100 O 1 5 100 O O 10 1O 90 1
A crude sample (135 pl) containing -28 pM AIASS-labelled C~S-~RNA~" was fractionated. Fractions that corresponded to the major peaks in the chromatograms were collected and assayed in the same manner as described for the fractionation of crude IAF-labelled C~S-~RNA~~~(vide ultra: section
2.3.3.1 ). After the final ethanol precipitation, each pellet was reconstituted in 10 pL of bufier A. Transfer RNA was monitored by urea-PAGE with a 10% 8 M urea/TBE polyacrylamide gel with ethidium bromide staining. Fluorescence from the AIASS rnoiety in AIASS-labelled C~S-~RNA~~'was monitored by fluorescence imaging.
A crude sample (135 PL) containing -28 pM IAEDANS-labelled Cys-
~RNA'" was fractionated and analyzed in the same rnanner as just described for
As controls, 10 PL of unacylated ~RNA'~'(-1 00 PM) and 10 pL of a crude sample that contained unlabelled C~S-~RNA~~'(-36%), disulphide-linked cysteinyl-~RNA'" (-4%), and unaminoacylated ~RNA'" (-60%) (- 40 pM total tRNA) were separately fractionated on the RX-C8 column using the solvent and gradient conditions just described for the fractionation of AIASS- and IAEDANS- modified ~~stein~l-t~~~~?Fractions that corresponded to the major peaks in the chromatograms were collected and assayed in the same manner as described for the fractionation of crude IAF-labelled C~S-~RNA~~.After the final ethanol precipitation, each pellet was reconstituted in 1O pL of buffer A, Nucleic acid was detected by urea-PAGE on a 10% 8 M ureaKBE polyacrylarnide gel with ethidium bromide staining.
After use, the RX-C8 column was washed with conditions specified in table 2.5 to remove NaCl. The column was stored in 80% methanol and 20%
H20-
Table 2.5- HPLC solvent and gradient conditions for removal of NaCi from the RX-C8 coIumn.
Tme (min) %A %B %C* Curve 2.4 Results and Discussion
In chapter 1. the cysteinyl side chain of C~S-~RNA~~~was labelled with a series of iodoacetarnide derivatives. Due to the partial charging of E- coli
~RNA'~'with cysteine by cysteinyl-tRNA synthetase and the incomplete reaction between cysteinyl-~RNA" and each of the labelling reagents, each of the labelled C~S-~RNA'~~constructs existed as a crude mixture consisting of approximately 60% unacylated ~RNA'~', 28% chemically-modified C~S-~RNA~~~,
6% unlabelled C~S-~RNA'~.and 6% disulphide-linked C~S-~RNA~~~.
Fractionation of the different components was not necessary for the binding experiments with GDP-bound and GTP-bound elongation factor Tu but is, however, significant for regeneration of unaminoacylated ~RNA'~and for the use of the chemically-modified C~S-~RNA~~'sto incorporate the chemically-modified amino acid into proteins (see chapter 3).
We synthesized four different chemically-modified C~S-~RNA~~constructs
(IAF-labelled C~S-~RNA'~.AIASS-labelled C~S-~RNA~~,IAEDANS-labelled Cys-
~RNA'", and BIADD-labelled C~S-~RNA~~(labels shown in chapter 1: figure
1-16) and employed reverse phase high performance liquid chromatography on an M-C8 column to fractionate the different components in each of the crude mixtures. 2.4.1 Characterization of chemically-modified C~S-~RNA~~~
Characterization of the fluorescently-labelled C~S-~RNA~~compounds
(IAF-, AIASS-, and IAEDANS-labelled C~S-~RNA~~~)were described in detail in chapter 1 (section 1.4.6).
BIADD-labelled C~S-~RNA~~was characterized by Northem blot (figure
2.3A (lane b)). A single band was detected which migrated at a position similar to that for biotinylated lysyl-t~~~LF(~ranscend~~ tRNA) (Promega) (structure shown in figure 2.38)(band observed in figure 2.3A (lane a). Unacylated
~RNA'" that-had been treated with the biotin iodoacetamide was analyzed on the same blot; however, no band was detected (figure 2.3A (iane c). In this way, it was confirmed that the cysteinyl side chah of C~S-~RNA~~~was successfully labelled with BIADD and no reaction of the label with the tRNA itself had occurred.
2.4.2 Fractionation of crude BIADD-labelled C~S-~RNA~~~and characterization of the peaks in the chromatogram
Crude BIADD-labelled C~S-~RNA~~~was chromatographed using the solvent and gradient conditions outlined in table 2.3. Figure 2.4 shows a representative chromatogram obtained. Peaks appeared at retention times (RT)
9 min, 10 min, il min, 18 min, 22 min, and between 40 and 45 min. The peaks labelled B-Pl (which comprises al1 sub-peaks from 8 - 12 min), B-P2, B-P3, and
B-P4 (which comprises al1 sub-peaks between the 40 and 45 min marks) were collected and analyzed by urea-PAGE with ethidiurn brornide staining
(henceforth, simply referred to as urea-PAGE). The results are shown in figure biotinylated aa-tRNA 3
Figure 2.3. (A) Northern blot of a 10% 8 M urea/TBE polyacrylamide gel showing Lys-tRNALYStagged with biotin (Transcende tRNA (control)) (lane a) and Cys-tRNACyStagged with biotin (BIADD-labelled Cys-tRNACyS) (lane b). Unacylated tRNACYStreated with the biotin labelling reagent BIADD was analyzed on the same blot, but no band was detected (lane c). Each band corresponds to -28 pmol of labelled tRNA. (B) TranscendTMtRNA structure (3' end only). mCf-- Y 2.5 A. The presence of biotin was monitored by Northern blot (figure 2.58). For comparison purposes, unaminoacylated ~RNA'~~and a crude sample containing unaminoacylated ~RNA'", unlabelled C~S-~RNA'",and disulphide-linked Cys-
~RNA'~'(sometimes referred to as crude cys-t~~ACFor just C~S-~RNA'" sample) were chrornatographed using the conditions from table 2.3 as well.
Their chromatograms are shown in figures 2.5 and 2.6, respectively. In the former chromatogram, peaks were observed at RTs 8 min, 9 min, and between
40 and 45 min (figure 2.5); whereas in the latter chromatogram, peaks appeared between 6 and 14 min and between 40 and 45 min (figure 2.6). The BU-PI (6 -
12 min) (short hand for combination of al1 sub-peaks between 6 and 12 min), BU-
P2 (40 - 45 min), BC-PI (6 - 14 min), and BC-P2 (40 - 45 min) peaks (figures 2.5 and 2.6) were collected and analyzed by urea-PAGE with ethidium bromide staining (figure 2.8).
The identity of some of the peaks in the chromatogram for BIADD-labelled
C~S-~RNA~~can be inferred.
- First, the peak with a retention time of 9 min (figure 2.4 (B-P1 (9 min))) is from unaminoacylated ~RNA'". This peak is also present in the chromatograrn for unaminoacylated ~RNA'~'(figure 2.5 (BU-P1 (9 min))). Urea-PAGE analysis revealed that fractions corresponding to the B-Pl (8 - 12 min) and BU-Pl (6 - 12 min) peaks contained nucleic acid with the same mobility as unacylated ~RNA'"
(figures 2.7A (lane b) and 2.8 (lane a)). Biotin was not detected in the B-Pl (8 -
12 min) fractions. disuiphide-lin ked CYS-tRNACy unacylated tRNACyS or Cys-tRNAQS (monomer)
disulphide-linked tCYS-tRNAQs biotinylated taa-tRNA
Figure 2.5. (A) Polaroid picture of an ethidium bromide-stained i0% 8 M urea/TB E polyacrylamide gel showing nucleic acid corn position in the fractions that cot-responded to the peaks in the HPLC chromatogram for crude BIADD-labelled Cys-tRNACYs(figure 2.4). Lane a: biotin-Lys-tRNALYS(control); lane b: fractions corresponding to the 8-Pi (8 - 12 min) peak; lane c: fractions corresponding to the B-P2 peak; lane d: fractions corresponding to the B-P3 peak; and lane e: fractions corresponding to the B-P4 (40 - 45 min) peak. (B) Western blot showing detection of biotin in TranscendTMtRNA (lane a) and the B-P2 (lane b) and B-P3 (lane c) fractions. No biotin was detected in the B-Pl (8 - 12 min) and B-P4 (40 - 45 min) fractions. The association of biotin with disulphide-linked Cys-tRNACySin lanes b and c appears to be due to non-specific binding of alkaline phosphatase- conjugated streptavidin (see section 2.4.2).
disutphide-linked Cys-tRNAQS
unacylated tRNACyS or ~ys-tRNAQS
figure 2.8. Polaroid picture of an ethidium bromide-stained 10% 8 M urea/TBE poIyacryiamide gel showing nucleic acid composition in the fractions that corresponded to the major peaks in the HPLC chromatograms for unarninoacylated tRNACYs(figure 2.6) (lanes a and b) and a c~desample containing unam inoacylated tRNACys,unlabelled Cys-tRNACYS,and disulphide-linked Cys-tRNACYS(figure 2.7) (lanes c and d). Lane a: fractions corresponding to the BU-Pl (6 - 12 min) peak; lane b: fractions corresponding to the BU-P2 (40 - 45 min) peak; lane c: fractions con-esponding to the BC-Pl (6 - 14 min) peak; and lane d: fractions corresponding to the BC-P2 (40 - 45 min) peak. The Peak at the 10 min mark (figure 2.4 (10 min))) anses from cystein~l-
~RNA~Y~.The chromatogram for unaminoacylated tRNACySdoes n0t contain this peak; however, it appears to be present in theœchromatogramfor C~S-~RNA~~~
(figure 2.7 (BC-PA (10 min))). The peak labelled BC-P1 is broad and appears to consist of several sub-peaks which are indistinguishable. Bands with the same electrophoretic mobility as C~S-~RNA~~~were observed in the urea-PAGE gels for the 8-Pl (8 - 12 min) and BC-Pl (6 - 14) fractions (figure 2.5A (lane b) and 2.8
(lane c)). Furthenore, bands which suggests the presence of disulphide-linked
C~S-~RNA~~~(formed by oxidation of the eluted monomeric C~S-~RNA~~~ molecules) were detected (figure 2.5A (lane b) and 2.8 (lane c)). Disulphide linked C~S-~RNA~~~that was fomed prior to chrornatographic separation eluted with a longer retention time than 10 min (vide infra). The presence of disulphide- iinked species was confirmed by treatment with TCEP, which caused a reduction in the intensity of the high molecular weight band and a proportional increase in the intensity of the low molecular weight band (data not shown). The relative intensities of the 9 min peak (which corresponds to unaminoacylated ~RNA'~') and 10 min peak (which corresponds to C~S-~RNA~~~)(cf. figures 2.4 (B-Pl (8 -
12 min)) and 2.7 (BC-Pl (6 - 14 min))) are different in the BIADD-C~S-~RNA~~~ and C~S-~RNA~~~chromtograms. In the former spectrum, the aminoacylated tRNA peak (10 min) is smaller than the unaminoacylated tRNA peak (9 min); whereas for the latter case, the two peaks are approximately the same in size.
The decrease in intensity of the C~S-~RNA~~~peak when going from the Cys- ~RNA'Y' to the labelled tRNA chromatograms is an indication that the cysteinyl side chain of the aminoacylated tRNA was successfully modified by BIADD.
The 11 min peak in the chrornatogram for BIADD-C~S-~RNA~~~(figure 2.4
(B-Pl (1 1 min))) is also observed in the chromatogram for C~S-~RNA~~'(figure
2.7 (BC-Pl (1 1 min))), but not in the chromatogram for unaminoacylated ~RNA'"; hence thiç peak also appears to correspond to C~S-~RNA~~~.This suggests the presence of two different species of C~S-~RNA~~~,which differ in hydrophobicity, in the B-Pl (8 - 12 min) and BC-Pl (6 - 14 min) fractions. This is consistent with the existence of two peaks with close retention times for unaminoacylated
~RNA'~'(figure 2.6 (BU-Pl (8 and 9 min))). There are several possible explanations for E. col; ~RNA'~'with two different retention times.
A logical explanation is that one species contains an unmodified adenosine residue at position 37 (non-native structure) and the other entity bears an msiA37 (msiA = 2-methylthio-N6-isopentenyl-adenosine) (chapter 1 : figure
1.8 D) (native structure). C~S-~RNA~Y~and ~RNA'~' with msiA37 are more hydrophobic than their respective counterparts without the modified base and therefore are expected to bind to the RX-C8 column with a stronger affÏnity. If this is the true, the peak with the longer retention time is assigned to the tRNA with the modified base. The modified bases of E. coli ~RNA~Y~(chapter 1: figures 1.7A and 1.8) are not considered to be identity determinants (i-e.features required for recognition of the tRNA by E. coli cysteinyl-tRNA synthetase) (see chapter 1 (section 1-1 -3.1)). This can be confirmed by an experiment in which an aminoacylation reaction with modified base-free ~RNA'~'is compared to that with the native tRNA. If the modified bases are indeed not important for the aminoacylation reaction, both species should be arninoacylated equally by the synthetase. Whether the ~RNA'~'stock used for Our experiments (obtained from
Subriden RNA) was of a single species or not remains to be determined. A potential method for finding out is to synthesize an E. col; ~RNA'~'which contains no modified bases (which can be achieved by in vitro transcription of the corresponding tRNA gene) and perforrning an HPLC experirnent using the exact same conditions as those employed for BIADD-labelled C~S-~RNA~~~.The chromatogram is than compared to the original.
The two different peaks for ~RNA~~'could also represent different conformational structures. For example, a loose structure is expected to bind tighter than a more compact structure due to differences in surface area (with the former species having the greater surface area). The transfer RNA adopts a tertiary structure (1-shape) that is governed by a series of tertiary base-base interactions (see chapter 1: figure 1.7). If a mutation occurs at one of the positions (or several mutations at multiple positions) that are involved in the folding of the tRNA (thus abolishing the native tertiary base-base interaction(s)) and not significant for ~RNA'Y' recognition by CysRS, a modified conformation
(from new tertiary base-base H-bonding) that is different from the native structure and still recognized by CysRS can theoretically arise. The tertiary base-base interactions that are identity elements for E. col; ~RNA'~'are G15:G48 and
A9:A13:A22 (see chapter 1 (section 1 .1.3.1)). Though the exact cause for two different retention times for the tRNA is at
present difficult to pin-point, sorne possibilities can be eliminated. For example,
mutations of the identity determinants (U73, anticodon GCA, G15:G48, and
A9:A13:A22) can be ruled out because both tRNA species can be
aminoacylated. Degradation is not a possibility since the degraded tRNA would
not have been recognized by the cysteinyl-tRNA synthetase. Thirdly, a cohrnn
with low binding efficiency (due to multiple uses) can be elirninated as well
because the RX-C8 used for our experirnents was new.
The peaks labelled B-P2 and B-P3 (retention times 18 and 22 min,
respectively) (figure 2.4) appear to correspond to BIADD-labelled C~S-~RNA~~'.
These peaks do not exist in the chromatograms for unaminoacylated ~RNA'Y'
and C~S-~RNA~~~.From the urea-PAGE experiments with the B-P2 and B-P3
fractions, bands with the same rnobility as biotinylated L~S-~RNA~~'(obtained
frorn Promega) were detected (figure 2.5A (lanes a, c, and d)). The Northern blot
reveals the existence of ~RNA~~~in association with a biotin tag with a molecular
- weight similar to that for biotin-~ys-t~~~~~~(figure 2-58 (lanes a, b, and c)). In
addition, the BIADD moiety makes the labelled tRNA more hydrophobic (hence a
longer retention time) than the unlabelled material. The two different retention
tirnes for BIADD-labelled C~S-~RNA'~~is in line with the existence of two different
species for unaminoacylated ~RNA'" and C~S-~RNA~~'juçt discussed (vide
ultra). Neither peak corresponds to unacylated ~RNA'~'labelled with biotin since
a chromatogram for unacylated tRNA that had been treated with BIADD (using
the same conditions as those employed for the biotinylation of C~S-~RNA'~')does not contain any peaks with retention times between 15 min and 30 min (data not
shown). Occurrence of a chemically-modified C~S-~RNA~~'construct in which
both the a-amino and thiol groups are modified by the biotin iodoacetamide
cannot be ruled out, however. The pK, of methyl S-rnethylcysteinate (methyl
ester of cysteine in which the side chain is methylated) has been reported to be
6.7 (10). By analogy, the acidity constant for the a-amino group in BIADD-
labelled C~S-~RNA~~~is expected to be similar. At pH 8.3, which is the pH
employed in our labelling reactions, the amino group should be deprotonated,
which makes it theoretically possible for the biotin iodoacetamide to react with
the nitrogen, thus forming a doubly-labelled C~S-~RNA~~~.Biotinylated Cys-
~RNA'" with two labels is expected to elute later than the monobiotinylated
species. One way of checking if such a reaction can occur is to react the methyl
ester of cysteine with BlADD using the conditions employed for the labelling of
C~S-~RNA~Y~(see section 2.3.1) (a reaction analogous to the synthesis of BIADD-
C~S-~RNA~~~).The products can be characterized by NMR. The Northern blot
for the B-P2 and B-P3 fractions also reveals a band which at first sight appears
to correspond to disulphide-linked C~S-~RNA~~*associated with biotin (i.e.
disuiphide-linked C~S-~RNA~~~in which the tRNA part itçelf is labelled with
BIADD). However, the chromatograrn for BIADD-treated unaminoacylated
- tRNACyS(not shown) is virtually identical to that for the untreated tRNA (figure
2-6); therefore, there was no non-specific labelling of ~RNA'~'. The OCCU~E~C~of
the high molecular weight bands is best explained by association of the
disulphide-linked C~S-~RNA~~~with alkaline phosphatase-conjugated streptavidin. Lastly, the series of sub-peaks detected between the 45 and 50 min mark in the BIADD-C~S-~RNA~~~chromatogram (figure 2.4 (B-P4 (40 - 45 min))) appear to arise from unaminoacylated ~RNA'~', ~~stein~l-t~~~~~~,disulphide- linked cys-t~~ACYS(fomed from oxidation of C~S-~RNA~~~before and af?er the fractionation), high molecular weig ht nucleic acid, and degraded nucleic acid fragments srnaller than ~RNA'~~(including degraded ~RNA~~'),as shown by the ethidiurn bromide-stained 10% 8 M ureamBE polyacrylarnide gel for the B-P4 (40
- 45 min) fractions (figure 2.5A (lane e). Biotin was not detected in these fractions. The same set of peaks is also present in the chromatograms for unaminoacylated tRNA (figure 2.6 (BU-P2 (40 - 45 min))) and cysteinyl-t~~~'~~
(figure 2.7 (BC-P2 (40 - 45 min))). The BU-P2 (40 - 45 min) peak corresponds to unacylated ~RNA'~"(figure 2.8 (lane b)); while the BC-P2 (40 - 45 min) signal appears to be due to unacylated ~RNA'~~,C~S-~RNA~~'. and oxidized Cys-
~RNA'~'(figure 2.8 (lane d)). The degradation of ~RNA'~'was not a result of
HPLC as it was observed in the original unacylated ~RNA'Y' stock (not shown).
The observations of three different retention times for unacylated ~RNA'~'(8, 9* and between 40 and 45 min) and C~S-~RNA~~~(10, 11, and between 40 and 45 min) are unexpected. ln the B-P4 (40 - 45 min) fractions, some of the unacylated tRNA may be a result of chemical hydrolysis of the C~S-~RNA'~~and disulphide- linked C~S-~RNA'Y~entities. The different states for ~RNA'~'can be accounted for by different conformational structures, as discussed above when the identities of the 9, 10, and 11 min peaks were assigned. We have shown that a crude sample containing BIADD-labelled Cys-
~RNA'~', unarninoacylated ~RNA'~',unlabelied C~S-~RNA~~~,and disulphide- linked C~S-~RNA~Y~can be fractionated by reverse phase liquid chromatography using an RX-C8 column. When the solvent and gradient conditions specified in table 2.3 were used, the labelled tRNA was isolated as a pure species.
Unacylated ~RNA'~',C~S-~RNA~~~, and disulphide-linked C~S-~RNA~~~,however. were not isolated as homogeneous entities. By experirnenting with shallower gradient conditions. however, al1 four tRNA constructs could probably be completely separated. For regeneration of unacylated ~RNA'~'and isolation of the chemically-modified entity for t~~~'~'-rnediatedmutagenesis (chapter 3), our results are sufficient because complets fractionation of the mixture of unacylated
~RNA~~'.C~S-~RNA'~~, and disulphide-linked C~S-~RNA~~'is not necessary.
2.4.3 Fractionation of crude IAF-labelled C~S-~RNA~~~and characterization of the peaks in the chromatograrn
Crude IAF-labelled C~S-~RNA~~~was chromatographed using the solvent and gradient conditions outlined in table 2.2. These conditions gave rise to the chromatogram displayed in figure 2.9. Peaks with retention times 9 min, 10 min, between 15 min and 20 min, 22 min, between 23 and 34 min, and between 40 and 45 min were observed. The absorbance from 23 - 34 min is a continuation of that between 15 min and 20 min (Le. a broad series of sub-peaks between 15 and 34 min). The fractions from the FI-PI (6 - 14 min), FI-P2 (15 - 20 min). FI-P3
(21 - 34 min). and FI-P4 (40 - 45 min) peaks were analyzed by urea-
PAGEIethidium bromide staining (figure 2.1 0A) and fluorescence imaging (figure FI-P 5
-0.05 IO 20 30 40 50 60 70 Retention tim (nlnute)
Figure 2.9. Typical chromatogram from fractionation of 100 pL of a crude sample that contained IAF-labelled Cys-tRNACYs(-28 pM) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions outlined in table 2.2. abcd abc d
dlsulphide-linked CystRNAW IA F-la belled dlsulphlde- llnked Cys-tRNAcr
unacylated or aminoacylated tRNACp
unbound fluoresceln
Figure 2.10. A) Polaroid picture of an ethidium bromide-stained 10% 8 M urea/TBE polyacrylamide gel showing nucleic acid content in the fractions corresponding to the peaks in the HPLC chromatogram for crude IAF-labelled Cys-tRNACYs(figure 2.9). Lane a: fractions corresponding to the FI-Pl (6 - 14 min) peak; lane b: fractions that corresponded to the FI-P2 (15 - 20 min) peak; lane c: fractions corresponding to the FI-P3 (21 - 34 min) peak; and lane d: fractions that corresponded to the FI-P4 (40 - 45 min) peak. (B) Fluorescence image of the unstained gel showing presence of fluorescein in the FI-Pl (6 - 14 min) (lane a), FI-P2 (15 - 20 min) (lane b), FI-P3 (21 - 34 min) (lane c), and FI-P4 (40 - 45 min) (lane d) fractions. 2.1 OB). Chromatograms were obtained for unaminoacylated ~RNA'~~(figure
2.1 1) and C~S-~RNA~~~(figure 2.1 2) samples as well. For the former situation, nucleic acid eluted at 9 min, between 15 min and 34 min, and between 40 min and 45 min; whereas in the latter case, peaks were observed at RTs 9 min, 10 min, between 15 min and 34 min, and between 40 and 45 min. The urea-PAGE results for the FU-P2 (15 - 34 min) and FC-Pl (7 - 13 min) fractions are shown in figure 2-13.
The 9 min peak in the IAF-C~S-~RNA~~chromatogram (figure 2.9 (F-Pl (9 min))), which is also observed in the chromatogram for unaminoacylated ~RNA'"
(figure 2.1 1 (FU-Pl (9 min))), is due to unaminoacylated ~RNA'~(as confirmed by the presence of unacylated ~RNA'" in the FI-Pl (6 - 14 min) (figure 2.10A
(lane a)) and FU-Pl (7 - 14 min) (data not shown) fractions.
The peak observed at the -IO min mark (figure 2.9 (FI-PI (10 min))) also exists in the C~S-~RNA~~chromatogram (figure 2.12 (FC-Pl (10 min))) but not in the unaminoacylated ~RNA'" spectrum. Nucleic acid entities with the same electrophoretic mobility as C~S-~RNA~"and disulphide linked C~S-~RNA~~
(formed from the eluted C~S-~RNA~~~)were detected in the FI-Pl (6 - 14 min) and
FC-Pl (7 - 14 min) fractions (figure 2.1 0A (lane a) and 2.13 (lane b), respectively). As in the situation with BIADD-labefled C~S-~RNA~~~(section
2-42),the shift in relative intensities of the 9 and 10 min singlets when going from the C~S-~RNA~~io the IAF-C~S-~RNA~" chromatogram (figures 2.1 2 and
2.9, respectively) is an indication that the cysteinyl side chain of the charged tRNA was derivatized by the IAF labelling reagent. The high molecular weight -0.003J Retention time (minute)
Figure 2.11. Typical chromatogram from fractionation of 25 pL of unaminoacylated tRNACyS(-100 FM) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions outlined in table 2.2.
disulphide-linked CYS-tRNACYS
unacylated tRNACYS or Cys-tRNACYS
Figure 2.13. Polaroid picture of an ethidium brom ide-stained 10% 8 M urea/TBE polyacrylamide gel showing nucleic acid content in the fractions that correspoonded to the major peaks in the chromatograms for unaminoacylated tRNACYS(figure 2.1 1) (lane a) and a crude çarnple containing unam inoacylated tRNACYS,unlabelled Cys-tRNACyS,and disulphide-linked Cys- tRNACYS(figure 2.12) (lane b). Lane a: fractions that corresponded to the FU-P2 (15 - 34 min) peak; and lane b: fractions from the FC-Pl (7 - 14 min) peak. nucleic acid in the FI-Pl (6 - 14 min) and FC-Pl (7 - 14 min) fractions (figures
2.1 OA (lane a) and 2.13 (lane b), respectively) corresponds to the tail-end of the
FI-Pl and FC-Pl series of sub-peaks which coincides with the beginning of the next series of peaks (figures 2.9 and 2.1 2) (vide infra). The same is true for the fluorescence detected in the FI-Pl (6 - 14 min) fractions (figure 2.1 OB (lane a)).
The broad peak from the 15 min mark to the 20 min mark (figure 2.9 (FI-
PZ (15 - 20 min))) (which is actually a multitude of sub-peaks) appears to arise from the elution of unarninoacylated ~RNA~~,c~s-~RNA~~~, disulphide-cys-
~RNA~",high molecular weight nucleic acid, and degraded nucleic acid fragments smaller than ~RNA~",as determined by the urea-PAGE analysis of the
FI-P2 (15 - 20 min) fractions (figure 2.10A (lane b)). The same broad set of peaks is also observed in the chromatograms for unaminoacylated ~RNA'"
(figures 2.1 1 (FU-PZ (1 5 - 34 min))) and C~S-~RNA~~(figure 2.12 (FC-P2 (15 -
34 min))). While the FU-P2 (15 - 34 min) fractions containeci unaminoacylated
~RNA'" (figure 2.1 3 (lane a)), the FC-P2 (15 - 34 min) fractions appeared to contain unacylated ~RNA'~', C~S-~RNA~~~,as well as disulphide-linked Cys-
~RNA'" (not shown). The occurrence of more than one retention time for unaminoacylated ~RNA'" and C~S-~RNA~"has already been discussed in the
BIADD-C~S-~RNA~~~experiment (vide ultra: section 2.4.2). The fluorescence image for the FI-P2 (15 - 20 min) fractions reveals the existence of IAF-labelled high MW nucleic acid, disulphide-linked C~S-~RNA'", C~S-~RNA~~,and unacylated ~RNA'" (figure 2.1 OB (iane b)). With the exception of IAF-Cys-
~RNA'" in which the label is attached to the cysteinyl side chain, the other fluorescent tRNA constructs must contain a dye molecule (or many dye molecules) attached to the tRNA. The elution of so many different tRNA constructs between 1: 5 and 20 min is consistent with the broadness of the relevant peak, which actually extends to the 34 min mark (see figures 2.9 and
2.12). The 4-thiouridine base (chapter 1: figure 1.8), which is present in the E. coli ~RNA'~'sequence (chapter 1: figure 1.7A), is known to be reactive towards
5-iodoacetamidofluoresceÏn and other iodoacetamides (1 1).
The FI-P3 (RT = 22 min) peak (figure 2.9) corresponds to IAF-labelled
C~S-~RNA~"(in which the dye is linked €0 only the cysteinyl side chain). This cornpound was detected in the FI-P3 (21 - 34 min) fractions via a combination of the 10% 8M urea/TBE polyacrylarnide gel and fluorescence image (figures 2.1 0A
(Iane c) and 2.1 OB (lane c), respectively). High MW nucleic acid (fluorescein-free and fluorescein-tagged), disulphide-linked C~S-~RNA~~(IAF-free and IAF- derivatized), unacylated ~RNA'~(fluorescein-free and fluorescein-bound) and low molecular weight fragments (IAF-free and IAF-labelled) were also observed, and to a greater extent than in the FI-P2 (15 - 20 min) fractions (cf. figures 2.1 OB, lanes b and c). The band migrating at the same position as IAF-modified Cys-
~RNA'" in the gel and Northern blot is by far the most intense, which is in line with the observation that the 22 min peak in the chromatogram is the most intense of al1 the sub-peaks between the 21 and 34 min retention times (see figure 2.9). This peak is not pure since it overlaps the other sub-peaks (figure
2.9). A distinct 22 min peak does not exist in the chrornatograms for unarninoacylated ~RNA'~'and C~S-~RNA~~.The sub-peaks of FI-P2 and FI-P3 are actually wnnected; this explains the similarity in nucleic acid composition between the FI-P2 (15 - 20 min) and FI-P3 (21 - 34 min) fractions.
The small peaks collectively designated as FI-P4 (RTs between 40 and 45 min) (figure 2.9) are also observed in the chromatograms for unacylated ~RNA'~' and C~S-~RNA~~~(figures 2.1 1 (FU-PB (40 - 45 min)) and 2.12 (FC-P3 (40 - 45 min)), respectively). The ethidium bromide-stained gel and the fluorescence image for the FI-P4 (40 - 45 min) fractions (figures 2-10A (lane d) and 2.1 OB
(lane d), respectively) reveals that the presence of unaminoacylated ~RNA'",
C~S-~RNA'~~,and IAF-labelled C~S-~RNA~~are possible , though the quantities are very low compared to those in the other fractions (Le. FLP2 (15 - 20 min) and
FLP3 (21 - 34 min)). An IAF-labelled C~S-~RNA~~species in which both the cysteinyl side chain and the or-amino group are modified would explain the long retention time for the Iabelled tRNA. This was discussed in section 2.4.4.
The fractionation of crude IAF-labelled C~S-~RNA~"using the solvent and gradient conditions outlilned in table 2.2 did not result in a complete separation of al1 the tRNA constructs; however, an IAF-labelled ~ys-t~~~~ysenrichedsample
(-90% enrichment) was obtained. For our purposes, this was sufficient.
2.4.4 Fractionation of crude AIASS-labelled and IAEDANS- labelled C~S-~RNA~~'and characterization of the peaks in the chromatograms
Crude AIASS-labeiled C~S-~RNA~~~was fractionated using the solvent and gradient conditions specified in table 2.4. lllustrated in figure 2.1 4 is a typical chromatogram from this experiment. Peaks were observed ai retention times 9 min, IOmin, 10.5 min, and between 30 and 35 min. When unaminoacylated - -
llll~~~~~~~~~~i~~~,~IlIll 10 20 30 40 50 60 Retention time (minute)
Figure 2.14. Typical chromatogram from fractionation of 50 pL of a crude sample that contained -28 FM AIASS-labelled Cys-tRNACYS(A) by RP-HPLC on an RX-C8 column using the solvent and gradient conditions specified in table 2.4. ~RNA'" was chromatographed, nucleic acids eluted at the 8.5 min, 9 min. and between 30 and 35 min marks (figure 2.1 5). Peaks with RTs between 7 min and
13 min, and between 30 and 35 min were observed in the experiment with Cys-
~RNA'~(figure 2.16).
Fractions from the S-Pl (8 - 14 min) group of peaks in the chromatogram for AIASS-labelled C~S-~RNA~F(figure 2.14) contained unacylated ~RNA'~,Cys-
~RNA'~,disulphide-lin ked C~S-~RNA~~,and AIASS-labelled C~S-~RNA~",as determined by urea-PAGE and fluorescence imaging (figures 2.1 7A (lane a) and
2.1 78). The 9 min peak is also obsewed in the chromatogram for unaminacylated ~RNA'~'(figure 2.1 5 (FU-Pl (9 min))) and therefore corresponds to unacylated ~RNA'~'(as confirmed by the presence of ~RNA~~'in the SU-Pl (8
- 14 min) fractions (data not shown)). Material that elutes at the 10 min mark is determined to consist of C~S-~RNA'" because the same peak also exists in the chromatogram for C~S-~RNA~~(figure 2.16 (SC-Pl (7 - 13 min))). Furthermore, disulphide-linked C~S-~RNA'~~,as well as a band that rnigrates with approxirnately the same molecular weight as C~S-~RNA~~~,were detected in the urea-polyacrylamide gel for the S-Pl (8 - 14 min) (figure 2.17A (lane a)) and SC-
Pl (7 - 13 min) (data not shown) fractions. The third sub-peak in the S-Pl group
(RT = 10.5 min) (figure 2.14 (S-Pl (10.5 min))) is assigned to AIASS-labelled
C~S-~RNA~";the reason being that the stilbene fluorophore makes the labelled tRNA more hydrophobic than unaminoacylated ~RNA~~'and C~S-~RNA~~'. This assignment is also confirmed by the fact that there is no 10.5 min peak in the other two chromatograms.
disulphide-lin ked Cys-t RNACYS j
unacylated or aminoacylated 3
Figure 2.17 A) Polaroid picture of an ethidium bromide-stained 10% 8 M urea/TBE polyacrylamide gel showing nucleic acid content in the fractions corresponding to the peaks in the HPLC chromatogram for crude AIASS- labelled Cys-tRNACYS(figure 2.14). Lane a: fractions corresponding to the S-Pl (8 - 14 min) peak; and lane b: fractions corresponding to the S-P2 (30 - 35 min). (0) Fluorescence image of the unstained gel showing presence of AIASS-labelled Cys-tRNACYSin the S-Pl (8 - 14 min) fractions. No fluorescence was detected in the S-P2 (30 - 35 min) fractions. Disulphide-linked C~S-~RNA~~.high molecular weight nucleic acid. as well as degraded nucleic acid from the AIASS-C~S-~RNA~~~sample eluted between
30 and 35 min (figure 2.14 (S-P2 (30 - 35 min))), as confirrned by urea-PAGE analysis of the S-P2 (30 - 35 min) fractions (figure 2.17 (lane b)). Unacylated
~RNA'" (and possibly C~S-~RNA~~~)were detected in these fractions as well.
The same set of peaks were observed in the chromatogram for unaminoacylated
~RNA'" (figure 2.1 5 (SU-P2 (30 - 35 min))) and C~S-~RNA'~(figure 2.1 6 (SC-P2
(30 - 35 min))). The SC-P2 (30 - 35 min) fractions appeared to contained Cys-
~RNA'~~.disulphide-linked C~S-~RNA'~,and unacylated ~RNA'~'(data not shown); whereas, rnostly unaminoacylated ~RNA'~~was found in the SU-P2 (30 -
35 min) fractions (data not shown). The AIASS fluorophore was not detected in the S-P2 (30 - 35 min) fractions.
IAEDANS-labelled C~S-~RNA~~~was chromatographed in the same manner as for AIASS-labelled C~S-~RNA'~~(conditions employed: table 2.4).
The chromatogram is shown in figure 2.1 8. Peaks were detected at retention times 9 min, 10 min, 10.5 min, and between 30 and 35 min. The results from urea-PAGE and fluorescence imaging analysis of the D-Pl (8 - 14 min) and D-P2
(30 - 35 min) fractions are shown in figure 2.19.
Using the same arguments as those used for the assignment of the peaks in the chromatogram for AIASS-labelled C~S-~RNA~~~,the D-Pl (8 - 14 min) and
D-P2 (30 - 35 min) peaks were identified. The 9 min, 10 min, and 10.5 min peaks correspond to unacylated ~RNA'", C~S-~RNA~~~.and IAEDANS-labelled
C~S-~RNA~~~,respectively. The peaks with retention times between 30 and 35
min appear to be from unacylated ~RNA'~', c~s-~RNA~~~,disulphide-linked Cys-
~RNA'", high-molecular weight nucleic acid, and degraded ~RNA'".
The conditions specified in table 2.4 allowed for removal of disulphide- linked C~S-~RNA~~~and some unacylated ~RNA'" from crude mixtures of AIASS- and IAEDANS-labelled ~ys-~RNA'? Cornplete separation of ~ys-~RNA'", labelled tRNA, unaminoacylated ~RNA~~,and disulphide-linked C~S-~RNA~~~was not possible with these conditions. A better separation could probably be achieved by experimenting with shallower gradient conditions than those given in table 2.4. This was nat further pursued, however, as the relatively low fluorescence of IAEDANS and AIASS (cornpared to IAF) made them less desirable for employment in t~~~'~'-rnediatedlabelling of proteins with fluorescent reporter groups (chapter 3). 2.5 Summary
The separation of unaminoacylated ~RNA'~,unlabelled ~~stein~l-t~~~~~~,
disulphide-linked C~S-~RNA~~~,and degraded ~RNA'" from labelled tRNA after a
chemical modification reaction between C~S-~RNA~~~and a thiol-reactive labelling
reagent is necessary for the use of the chemically-modified C~S-~RNA~~in
t~~~'~-rnediatedprotein labelling (see chapter 3). Furthermore, it allows for
regeneration of ~RNA'" for other experiments. We attempted to develop solvent
and gradient conditions for the RP-HPLC (with an RX-C8 column) purification of
BIADD-, IAF-, IAEDANS-, and AIASS-labelled C~S-~RNA~~(see chapter 1 :
figure 1.16a - d for chemical structures).
We were able to develop conditions for the purification of BIADD-Cys-
~RNA'" (table 2.3).
When the mixture containing IAF-C~S-~RNA~~~was chromatographed
using the conditions outlined in table 2.2, the different tRNA components were
not resolved as effectively as in the BlADD experiment; a 90% enrichment was
achieved. The enriched Iabelled tRNA was found to be "contaminated" with
unacylated ~RNA~",fluorescein-tagged unacylated ~RNA'~,C~S-~RNA~~~,
disulphide-linked C~S-~RNA~~~,IAF-labelled disuiphide-linked C~S-~RNA~~~,
degraded ~RNA~",and high molecular weight nucleic acid (fluorescein free- and fluorescein-bound). For improvement, a shallower gradient in the region where the labelled tRNA elutes should be employed and the composition of solution A should be changed so that the concentration of salt is much lower. The tRNA components that are non-specifically labelled can be eliminated by the use of labelling conditions which are milder than those in our experiments (chapter 1
(section 1.3.1 O)). The alternative is to protect the 4-thiouridine at position 8
(chapter 1 (tigure 1.7A)) (discussed in chapter 3 (section 3.7.1)). Lastly, a centrifuga1 filtration device equipped with the appropriate molecular weight cut-off porous membrane should allow removal of the degraded tRNA and high MW nucleic acid.
For AIASS- and IAEDANS-modified C~S-~RNA~~~,the separation was the worst. The conditions employed are shown in table 2.4. Basically, we were only able to remove the disulphide-linked C~S-~RNA~~~material. Further purification can be achieved by deliberately oxidizing the C~S-~RNA~~~component in the S-
PI (8 - I4 min) and D-P 1 (8 - 14 min) (figures 2.14 and 2.1 8, respectively) fractions (with mxygen) in the enriched samples and rechromatographing. A shallower solvent gradient in the region where the labelled tRNA elutes should provide a better resolution. The AIASS and IAEDANS fluorophores exhibit very weak fluorescence relative to fluorescein; for this reason, they were not further used in other experiments (chapter 3).
Lastly, we have shown that RP-HPLC is useful even for the resolution of large entities (MW -25 000) which have small structural differences (e-g. unacylated ~RNA'~'versus C~S-~RNA~~). 2.6 Reference
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2. Krieg, U. C., Walter, P., and Johnson, A E. (1986) Photocrosslinking of the
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3. Wiedmann, M., Kurzchalia, T. V., Bielka, H., and Rapoport, T. A. (1987)
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4. Kurzchalia, T. V., Wiedmann, M., Breter, H., Zimmermann, W., Bauschke, E.,
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5. Crowley, K. S., Reinhart, G. D., and Johnson, A. E. (1993) The signai
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6. Kabat, D., Hoffman, B., and Rich, A. (1970) Synthesis and characterization of
a spin-labelled aminoacyl transfer ribonucleic acid. Biopolymers 9, 95-1 01 7. Gillam, I., Millward, S., Blew, D., Tigerstrom, M. v., Wimmer, E., and Tener,
G. M. (1967) The separation of soluble ribonucleic acids on benzoylated
diethylaminoethylcellulose. Biochemistry 6, 3043-3056
8. Odom, O. W., Kudlicki, W., and Hardesty, B. (1998) In vitro engineering using
acyl-derivatized tRNAs. In Profein Synthesis Methods and Protocois pp. 93-
103, Humana Press, New Jersey
9. Gite, S., Mamaev, S., Olejnik, J., and Rothschild, K. (2000) Ultrasensitive
fluorescence-based detection of nascent proteins in gels. Anal Biochem 279,
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10. Hay, R. W., and Porter, L. J. (1967) Proton ionisation constants and kinetics
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chernical labe[ling of DNA fragments. Nucleic Acids Res 7, 7 485-1495 Chapter 3
Towards ~RNA'~*-M~~iated Labelling of Firefly Luciferase with Non-Natural Reporter Groups Introduction
3.1.1 Incorporation of fluorescent non-natural amino acids into proteins (applications)
Proteins with fluorescence reporter groups can be characterized via
fluorescence spectroscopy. In particular, its structure, cellular localization, and
dynamics can be investigated (1).
Fluorescence ernission intensity and wavelength are affected by the local
environment of the Ruorophore. For example, emission from a surface tryptophan (Trp) residue which is exposed to the aqueous solution occurs at
longer wavelengths than that from a Trp that is buried in the interior of a protein
(1). Extrinsic probes (Le. unnatural fluorophores) can be used to determine a probe's location on a protein. For instance, the dye 6-(p-toluidiny1)naphthalene-
2-sulfonic acid (TNS) is weakly fluorescent in water; however, upon addition of apomyoglobin to a solution of the fluorophore, the TNS fluorescence intensity increases and emission spectrum shifts to shorter wavelengths (1). The changes
in the fluorescent properties of the reporter group reflects the hydrophobie nature of the protein's heme binding site.
Fluorescence quenching is a phenomenon whereby fluorescence intensity is decreased by molecules and/or ions known as quenchers. Some commonly used quenchers are iodide (1-), oxygen, and acrylamide (1). The accessibility of fluorescent molecules to such quenchers can be used to determine the location of probes on a protein, or the porosity of proteins to quenchers. For instance, the emission intensity of a tryptophan located on the surface of a protein decreases in the presence of iodide (1). Conversely, if Trp were buried inside a protein, accessibility to the fluorophore by the I- ions would be reduced (1 ).
Fluorophores absort, light along a particular direction with respect to the moIecular axes (1). The extent to which a fluorophore rotates during the excited state lifetime determines its polarization or anisotropy (Le. degree of scrambling of the incident polarized Iight) (1). The more rigid the attached probe, or the slower the attached probe rotates, the higher the polarization of the emitted light.
The phenomenon of fluorescence anisotropy has been employed to determine the apparent volume, or molecular weight, of proteins, which is possible because larger proteins rotate more slowly (1 ). Hence, if a protein associates with another protein, the rotational rate decreases while the polarization increases. This cm be employed to monitor protein-protein interactions (1 ).
Fluorescence resonance energy transfer (FRET), which is a phenomenon whereby the excitation of a fluorophore (donor) leads to the excitation of a second nearby fluorophore (acceptor), provides an opportunity to measure the distance between sites on a protein (1). Measured transfer efficiencies can be used to calculate the distance between the two relevant sites (1 ). FRET may also be employed to monitor protein-protein interactions (1).
Tryptophan is the only naturally-occuning amino acid that is fluorescent.
The indole group absorbs at 280 nm and emits near 340 nrn, and has an emission spectrum that is highly sensitive to solvent polarity (1 ). The utility of Trp for fiuorescence studies is often hampered by the presence of other tryptophans in the same protein (2). To solve this problem and also for the utilization of other fluorescent molecules, it is important to deveiop general methods for biosynthetic incorporation of fluorescent non-natural amino acids.
3.1.2 Tech niques for biosynthetic incorporation of non-natural amino acids into proteins (in vitro)
Site-directed mutagenesis is a technique whereby a native arnino acid at a specific position in a protein can be replaced with another residue. The mutation occurs at the gene Ievel whereby the codon for the original amino acid is simply replaced by a new triplet that specifies for the new amino acid. The technique is limited by the feature that a particular amino acid can only be mutated to one of the other 19 naturally encoded residues.
The most widely used method for introducing labels into proteins is post- translational (Le. after the protein has been biosynthetically synthesized and folded into the native structure) chemical modification of the amino acid side chainç (3). For example, side chains such as the mercaptomethyl group of cysteine and the aminobutyl moiety of lysine can be selectively chemically modified with reagents which are thiol- and amino-reactive, respectively. A general list of such labelling reagents is shown in chapter 1 (schemes 1.1A and
1.1 C) (4).
Chemical derivatization of proteins in solution has often been criticized as being problematic for several reasons. For instance, experimenters often have to contend with the issue of site-specificity. A thiol-reactive compound will, in general, react with al1 accessible cysteine residues, while amino-reactive chemicals will generally derivatize ail lysine residues. Although cysteine is relatively rare (-1 -7% (5)),lysine, on average, represents -6.6% of a protein's amino acid content (Prornega technical bulletin TB182). Furthermore, reaction conditions must be fine-tuned to ensure that just one type of side chain is being modified. For example, harsh conditions (such as extreme alkalinic pH (which causes both the cysteinyl and lysyl side chains to bec0m.e deprotonated), high temperature, and long incubation time) may result in modification of both the cysteinyl and lysyl side chains by certain labelling reagents (e-g. iodoacetamide- containing compounds) (4). Incorporation of multiple labels into a protein can complicate subsequent measurements. The site-specificity problem escalates as the target protein increases in size because there are more side chains to consider. Another potential difficulty with post-translational labelling is that side chains that are buried in the interior of a globular protein are inaccessible by the labelling reagent. Protein denaturation, which is required to gain access to the interior residues, should be avoided because it is often irreversible or chaperones are required for regeneration of the protein native structure.
Stepwise solid-phase peptide synthesis (the chemistry for which is not elaborated here) (6) is another method for incorporating unnaturai amino acids
(7, 8). This is a powerful strategy as virtuaily any non-cognate residue can be introduced during synthesis of the peptide. However, the chemically synthesized peptides are generally restricted to having short sequenoes (50 - 60 residues) due to statistical accumulation of resin-bound byproducts (which hampers purification) (9). Furthermore, large proteins are generall y accompanied by low yields and long reaction times. The problem with protein size can be overcome by cornbining solid-phase peptide synthesis with a procedure called thioester-mediated native chemical
Iigation (9). The Iigation method (the chemistry for which is not described here), involves chemically joining two or more polypeptide segments (synthesized by solid-phase chemistry) together (9)- For the ligation of multiple peptides, usually a solid-phase-based approach is employed (9). In this manner, synthesis of polypeptide chains of up to eight segments has been demonstrated (9). The solid-phase Iigation protocol can also be extended to ligate large polypeptide segments (Le. > 60 amino acids) produced by recombinanat-DNA-based expression in genetically engineered microorganisms (referred to as expressed protein Iigation) (9). The ability to mix-and-match recombinant and synthetic polypeptides has made it possible to introduce unnatural amino acids containing biophysical probes into large proteins (10-1 2).
Despite the recent improvements in the total chemical synthesis of proteins, the procedure is still inhibited by the fact that the large membrane- bound proteins tend to be difficult to produce.
If the aforementioned drawbacks for protein labelling by post-translatioinal chemical modification and total chemical protein synthesis are to be circumvented, a technique is required which makes possible incorporation of non-naturcil amino acids during in vitro or in vivo ribosome-based protein biosynthesis. 3.1.2.1 The classic Raney-Nickel experiment
The development of biosynthetic approaches for incorporation of non- standard amino acids into proteins was pioneered by a classic experiment that was conducted in the early 1960s by Chapeville and colleagues (13). The goal the researchers had in mind was to prove the "Adaptor Hypothesis", that tRNA molecules contain one end (the acceptor moiety) to which the cognate amino acid is appended and a second end (the anticodon domain, which is Iocated at the opposite end of the tRNA relative to the acceptor section) which is responsible for recognizing the codon that codes for the amino acid. The implication of the "adaptor hypothesis" is that tRNA moIecules play the role of ensuring that the amino acid residues in a protein are joined in the predetermined order as specified by the order of codons in the corresponding mRNA template
(14, 15). Specifically, Chapeville and collaborators wanted to prove that the coding property of the ~~çteinyl-t~~~~~construct iç determined by the ~RNA~~~,AI~-~RNA~" (alanyl-t~~~A'a), and AI~-~RNA'" were used in three separate in vitro translation reactions that were programmed with poly (UG) (Le. UGUGUG... ) (an mRNA with codons (UGU) that encode for polycysteine) and were capable of protein synthesis. The translation reaction with C~S-~RNA~" gave rise to polycysteine polypeptides; the experiment with AI~-~RNA~'=did not C ysleine if I I 0- C-C-C-SH Cystcine Acccpior sRNt Il Cysleine 1Raney Nickel Alanine Scheme 3.1 . Desulfhydration of 1-cysteinyl-tFINAqSwith Raney-Nickell produces L-alanyl-tRNACyS. (Diagram obtained from Chapeville et al. publication (13)). produce any poly (UG)-directed peptides; finally. A~~-~RNA~~'resulted in the synthesis of polyalanine products. The observation that AI~-~RNA~~~,but not Ala- ~RNA*'~,was accepted by the poly (UG)-programmed ribosome led to the conclusion that the tRNA moiety, not the aminoacyl group, determines the coding specificity of aminoacyl-tRNA. From the point of view of non-natural amino acid mutagenesis (NNAAM), this was the first demonstration that an amino acid could be biosynthetically incorporated into a peptide by supplementing an in vitro translation reaction with an aminoacyl-tRNA whose aminoacyl side chain had been chemically modified. In a related experiment, Chapeville and CO-workersshowed that the cysteine in cysteinyl-~RNA~"may also be oxidized to cysteic acid (scherne 3.2), a rather drastic change in the chernical properties of the amino acid, without loss of reactivity with poly (UG) in a translation assay (16). This was the first report of incorporation of an unnatural amino acid into a polypeptide by the ribosome- based translation process. In 1963, von Ehrenstein and co-workers showed that the misincorporation of alanines into the polypeptide encoded by the artifical messenger RNA poly (UG) via the employment of the hybrid a1an~1-t~~~~~construct could be extended to translation experiments in which the mRNA template directed the biosynthesis of hemoglobin (a natural protein) (17). Three translation experiments using rabbit reticulocyle lysates (RRL) were performed. Each translation system was programmed with the mRNA for hemogiobin and supplemented with one of the E. coli aa-tRNA constructs AI~-~RNA*",Ala- ~RNA'", and cys-t~~ACF.It was found that alanine from AI~-~RNA*"was incorporated at sites which were norrnally occupied by alanine residues, cysteine frorn C~S-~RNA~"was detected to occur at positions which were normally represented by cysteine, and alanine from the hybrid AI~-~RNA~~construct was incorporated into sites which, under native conditions, were occupied by cysteine and not alanine- This confirrned the result that the tRNA molecule, rather than the amino acid, is responsible for the coding property of aminoacyt-tRNA. 3.1.2.2 Chemical modification of the a-amino group in aminoacyLtRNA A research group led by Hardesty introduced a technique for incorporating non-natural amino acids into proteins that involves the use of N-acylaminoacyl- tRNA derivatives (aminoacyl-tRNAs in which the cc-amino group of the aminoacyl rnoiety has been acylated with a small molecule) (1 8). The entire technique is outlined in scheme 3.3 for the use of coumarin-labelled M~~-~RNA~~'(19). The first step involves preparation of the synthetic aminoacyl-tRNA CPM-SAc-Met- ~RNA~~~.The initiator ~RNA~~'from E. coli is aminoacylated with methionine using methionyl-tRNA synthetase (MetRS), as the catalyst, and ATP as a cofactor. In a separate reaction, DTDG (dithiodiglycolic acid = the disulphide of mercaptoacetic acid) is converted to its monosuccinimidyl ester counter part by coupling with N-hydroxysuccinimide. When the product of the aminoacylation reaction, methionyl-~RNA~~',is reacted with the DTDG monosuccinirnidyl ester, an ~-acylmethion~l-t~~~~derivative containing a disulphide is synthesized. Upon treatment with DTT, the mixed disulphide is reduced, and an N- acylmethionyl-~RNA~~'construct with a free thiol group is generated. The CPM- w 2) DTT E. col/ cell-free coupled transcriptionltranslation system programmed with the gene of interest Protein labelled wiih CPM-SAc-methionine at the N-terminus Scheme 3.3. Strategy for incorporation of a coumarin-labelled methionine amino acid at the N-terminus of proteins by the use of CPM-SAc-Met-tRNAMe'. SAC-M~~-~RNA~~'adduct is finally constructed when the thiol group of the latter species is akylated with CPM (7-diethylarnino-3-(4'-maleimidylphenyl)-4- methylcoumarin). CPM-SAC-M~~-~RNA~"is then isolated as a purifed species by HPLC. The fluorophore is incorporated into a protein by means of a coupled transcription/translation reaction using an E. coli cell-free protein synthesis system programmed with the gene of interest and supplemented with CPM-SAC- M~~-~RNA~"'. The synthetic tRNA constructs (with their acylated amino groups) in this technique can only serve as donors in protein synthesis and thus can only be incorporated at the N-terminus of a target protein. A protocol closely related to that shown in scherne 3.3 has been used by several investigators to incorporate the coumarin fluorophore into the N-terminal end of polypeptides polyphenylalanine, polyalanine, polyserine, polycysteine, and polylysine (20-22).In a similar manner, the AEDANS fluorophore (figure 3.1 ) has been incorporated into polyphenylalanine and polylysine via the use of AEDANS- SAC-M~~-~RNA~~~(21 ). In al1 these experiments, the immobilized fluorescent probes were used to study interactions and the environment of the nascent polypeptide chains in the 50s ribosomal subunit during chain elongation during the synthesis of the polypeptides. Fluorescence anisotropy was employed to monitor the rigidity of the fluorophore as the N-terminus moved away from the peptidyltransferase center. The polarity or hydrophobicity of the surrounding milieu was measured by changes in the fluorescence intensity and wavelength of maximum emission. The accessibility of the nascent polypeptide chains to the cytoplasrnic environment was investigated by collisional quenching assays. In another example, the sequence of reactions outlined in scherne 3.3 was employed by Kudlicki and colleagues to incorporate the coumarin dye (figure 3.1) at the N-terminus of rhodanese (18). The fi uorescence properties of the tethered coumarin rnolecule were exploited to study the mechanism of rhodanese folding. in a very recent publication, Gite and colleagues described the use of BODIPY-FL-~~~~~O~~~-~RNA~~~(BODIPY-FL = 4,4-difluoro-5,7-dimethyl4-bora- 3a,4a-diaza-s-indacene propionic acid) (figure 3.1 ) as a tool for fluorescence- based detection of in vitro synthesized proteins (23). The BODIPY-FL dye was successfully incorporated into a-hernolysin, dihydrofolate reductase, luciferase, chloramphenicol acetyltrasnferase, and bacteriorhodopson (23). 3.1.2.3 Chernical modification of the &-aminogroup in lysyl-t~~~LYs Site-specific labelling of proteins by the use of chemically modified lysyl- ~RNA~~'constructs is illustrated in scherne 3.4 (24). Yeast ~RNA~~'is aminoacylated with lysine in an aminoacylation reaction with yeast lysyl-tRNA synthetase (LysRS) and ATP. The &-amino group of the lysyl side chain in Lys- ~RNA~~'is then modified with a reagent that contains a succinimidyl ester moiety. BD cellulose chromatography is then employed to purify the modified tRNA. For incorporation of the unnatural amino acid acylated to ~RNA~~'into proteins, the synthetic tRNA construct is added to an in vitro wheat germ cell-free protein synthesizing system that has been programmed with the required gene. The unnatural amino acid is incorporated at positions which are normally occupied by IL) IL) -* al O E 8 01 unmodified lysine. This method overcomes the limitation imposed by the use of N-acylaminoacyl-tRNA in that unnaturaI residues could be introduced at positions other than the N-terminus. This type of non-natural amino acid mutagenesis has been employed to label preprolactin (a secretory protein from the pituitary gland) with several different photoafinity groups. The photo-reactive probes that have been successfully incorporated are p-azidobenzoic acid (ABA) (24), 5-azido-2- nitrobenzoic acid (ANB (25), and 4-(3-trifluoromethyldiazirino)benzoic acid (TDBA) (26) (chapter 2: figure 2.1A, BI and CI respectively) (via the use of E- ABA-L~s-~RNA~",E-AN B-L~S-~RNA~~~, and E-TD BA-L~S-~RNA~~~, respective1 y). Several investigators have used an incorporated photoaffinity label to detect protein-protein interactions between the nascent chain of preprolactin and adjacent proteins during translation and translocation across the endoplasmic reticulum (24-32)- Upon photolysis, the Iabel formed a cross-link with adjacent proteins. Kurzchalia and colleagues have provided data to show that a chemically- modified L~S-~RNA~"construct in which the e-amino group is tagged with a biotin derivative could be used as a tool for non-radioactive detection of in vitro translated proteins (33). They were able to incorporate unmodified biotin as well as N-biotinyl-6-aminohexanoic acid (AhxBio) via the use of biotin-labelled Lys- ~RNA~~(E-B~O-L~S-~RNA~~) and AhxBio-labelled L~S-~RNA~~~(&-AhxBio-Lys- ~RNA'~')(chapter 2: figures 2.1 D and E, respectively). The latter semi-synthetic tRNA is now commercially available from Promega. Finally, Crowiey et al. (34) as well as Harnman and collaborators have successfully incorporated a fluorescent non-standard amino acid into the signal sequence of preprolactin (34, 35). The fluorophore is 6-(7-nitrobenz-2-oxa-1,3- diazol4-y1)aminohexanoic acid (NBDAhx) (chapter 2: figure 2.1 F) and was delivered from NB DAhx-labelled (NB DA~X-L~S-~RNA~~~).Crowl ey and CO-workersused fluorescence life-tirne and collisional quenchers to characterize nascent preprolactin's exposure to the cytoplasrn and environment at the endoplasmic reticulum. In the experiments conducted by Harnrnan and colleagues, fluorescent dyes inwrporated at specific sites along nascent preprolactin polypeptides were used in collisional quenching assays to determine the intemal diameter of a functioning protein translocation pore in the endoplasmic reticulumn. This was the first and is the only report of the use of chemically-modified L~S-~RNA~"for the incorporation of fluorescent non-standard amino acids into proteins. 3.1.2.4 Non-sense suppression The rnost widely used technique for site specific protein iabelling is non- natural arnino acid mutagenesis via amber suppression. The overall methodology is outlined in scheme 3.5 (36). This method was developed in the labs headed by Charnberlin (371, Schultz (38),and Hecht (39). A nonsense (stop) codon is introduced at the site of interest in the gene of interest using conventional site-directed mutagenesis. A nonsense codon, under normal circumstances, functions as a signal for termination of protein translation. Chain termination is assisted by proteins called release factors that recognize the Codon for residue of interest Nonsense codon AGC TAG Y Y T4 RNA ligase transcription In vitro translation f Mutant enzyme with unnaturai amino acid site-specifically Suppressor tEWA (-CA) incorporated. Scheme 3.5. Outline of non-natural amino acid rnutagenesis by amber suppression. (Illustration obtained from Ellman et al. PaPer (36))- temination codon (40). In vivo, there are normally no tRNAs that are able to translate a temination codon. Three different stop codons exist: amber (UAG), ochre (UAA), and opal (UGA). The triplet most cornmonly employed in NNAAM is UAG. UAG was the codon of choice because amber suppressors consisting of mutant tRNAs with CUA anticodons had long been known to insert amino acids at premature UAG codons in mRNA without arresting cell growth (41). By introducing a UAG codon at a premature position in the open reading frame of a gene, the gene has two stop codons. The UAG is for NNAAM, while the second termination codon, which is not UAG but rather either opal or ochre, occurs at the end of the gene and serves to terminate chain elongation during protein synthesis. In a separate process, a semisynthetic aminoacyl suppressor tRNA which contains a CUA (anti-amber) aniticodon and an unnatural aminoacyl side chain is constructed. The anticodon is capable of translating the UAG message on the mRNA. When the gene, in the forrn of a plasmid, and the aminoacyl suppressor tRNA are combined in an E. coli cell-free coupled transcriptionltranslation system, the unnatural amino acid delivered by the suppressor tRNA (if accepted by the protein biosynthetic machinery) is inserted into the protein at the site that corresponds to the UAG codon. Full-length proteins are produced if the amber codons were suppressed; however, no suppression gives rise to truncated proteins. This provides a means of determining whether an unnatural amino acid is incorporated or not. Several critical features of the aminoacyl suppressor tRNA shouid be mentioned. First, the suppressor tRNA is designed so that it is not recognized by any of the endogenous aminoacyl-tRNA synthetases (aaRS). Such a tRNA is often referred to as an orthogonal suppressor tRNA (O-tRNA). The orthogonality is necessary to avoid aminoacylation of deacylated suppressor tRNA with an undesired endogenous amino acid and the subsequent incoporation of residues other than the unnatural amino acid. The use of a non-cognate suppressor tRNA also prevents enzymatic deacylation of the aminoacyl suppressor tRNA. An orthogonal suppressor tRNA was created by mutating base pairs in the original tRNA so as to remove recognition elements that are important for binding of the native tRNA to the wgnate aminoacyl-tRNA synthetase (42). Synthesis of the arninoacyl suppressor tRNA is complicated and labor- intensive. An outfine for construction of an aminoacyl suppressor tRNA based on E. coE ~RNA~'~~is shown in scheme 3. 6 (42). The procedure was originally developed by Roesser and co-workers (43). Synthesis of the aminoacyl suppressor tRNA itself occurs in three stages: synthesis of the arninoacylated pdCpA dinucleotide; synthesis of the 3' truncated suppressor tRNA in which the terminal CA nucleotides have been removed; and ligation of the aminoacylated dinucleotide and truncated suppressor tRNA. The 3'-truncated suppressor tRNA is typically synthesized by run-off transcription using a Iinearized plasmid carrying the tRNA gene, and 77RNA polymerase (42). The T7 transcript has no hypermodified bases; however, it has been shown that the hypermodifications are not necessary for the suppressor tRNA to be functional (37, 44). The truncated suppressor tRNA and the aminoacylated dinucleotide are ligated using T4 RNA Iigase (scheme 3.6). The principle advantage of NNAAM by amber suppression is the ability to site-specifically label a protein at a single position, which is not possible with post-translation chemical modification or via chemically-modified L~S-~RNA~? Labelling at multiple sites is undesirable because it can complicate subsequent measurements. Though conventional site-directed mutagenesis could be utilized to replace excess lysine residues with a similar amino acid, such as arginine, and hence engineer a protein with a single lysine amino acid, removal of several critical lysines could be detrimental to protein stability and hence function. Arnber suppression non-natural amino acid mutagenesis has made possible the incorporation of residues containing novel structural, chemical, and spectroscopic properties. A list of unnatural amino acids that have been successfully incorporated is shown in figures 3.2 and 3.3 with the fluorescent residues indicated (36, 45-48). These amino acids have been used by various investigators for studies on protein structure (49-51 ), protein-protein interactions (47, 52), enzymatic catalysis (53),and molecular recognition (54). 3.1.2.5 Frame-shift suppression The frame-shift suppression strategy was developed by Hohsaka and colleagues (55). The idea is similar to that for amber suppression (scheme 3.5). The codon of interest is replaced with a four-base codon (AGGU, AGGG, AGGC, or AGGA) instead of a termination codon using conventionial site-directed mutagenesis. In a separate process, an aminoacyl-tRNA with an unnatural aminoacyl side chain and a four-base anticodon (ACCU, CCCU, GCCU, or UCCU) that is complementary to the four-base codon is constructed (as oppose to an anti-amber anticodon). Addition of the mutagenized gene, in the form of a plaçmid, and the synthetic tRNA construct to an E. co/i S30 extract coupled transcription/translation system results in introduction of the unnatural amino acid into the protein at the position that corresponds to the four-base mutation. The aminoacyl frame-shift suppressor tRNA was synthesized in three stages in a similar manner as that described for the preparation of the aminoacyl amber suppressor tRNA (vide ultra: section 3.1 -2.4 (scheme 3.6)) (55). The synthetic tRNA was derived from yeast ~RNA'~~.Like the anti-amber tRNA, the frame-shift suppressor tRNA is not recognized by any E. mli endogenous arninoacyl-tRNA synthetases. The mutagenized gene encoding the protein under study is designed such that full-length proteins are produced only when frame-shift suppression by the synthetic aminoacyl-tRNA occurs. Truncated proteins are observed when the four-base codon is decoded as a triplet by E. coii endogenous tRNA such as ~RNA~'~.An example is shown in scheme 3.7 for a mutated streptavidin (55). A consequence of this is that only full-length proteins that are translated from the mRNA template contain the unnatural amino acid. This provides a simple method for detecting incorporation of the unnatural amino acid. Arg XaaT Val Leu Xaa* Gly ~rg'.Tyr Asp \' / Scheme 3.7. Frame-shift suppression versus in-frame translation in frame-shift suppression mutagenesis. Nucleotide and amino acid sequence of a mutated streptavidin. Underlines indicate stop codons which appear when one of the four-base codons is decoded as a triplet by E. coli endogenous arginyl tRNA. (Diagram obtained from Hohsaka et al. publication (55)). In the aforernentioned NNAAM methodofogy in which a termination codon is employed, the suppression by the arnber suppressor tRNA competes with a termination of protein synthesis by release factors. Furthermore, only two of ihree termination codons are available for introducing non-standard residues; hence, a maximum of two different non-natural amino acids can theoretically be concurrently introduced into different sites in the same protein, though this has not been reported. These [imitations do not exist for the frame-shift suppression methodology. In fact, Hohsaka and colleagues have reported simultaneous introduction of two different non-natural amino acids into streptavidin (56). Using this technique, the protein streptavidin has been labelled with a variety of flurophores and other aromatic cornpounds (figure 3.4) (56-58).These nonnatural amino acids have been used to study the binding affinity between streptavidin and biotin (58, 59) and the adaptability of aromatic nonnatural amino acids to the ribosome in E. coli and the rabbit reticulocyte (vide intra: section 3.1 -4.2) (57)- 3.1.3 Techniques for biosynthetic incorporation of non-natural amino acids into proteins (in vivo) 3.1.3.1 In vivo incorporation by injecting synthetic aminoacyl-tRNAs into Xenopus laevis oocytes Membrane-bound proteins, such as receptors, channels, and transporters, are an important class of proteins but, because of their size and environmental requirements, they have been difficult to characterize structurally using the traditional NMR and X-ray crystallography methods. Non-natural amino acids -ch- incorporated at specific positions in these proteins in intact Xenopus laevis oocyte cells via the use of aminoacyl amber suppressor tRNAs makes possible employment of non-traditional techniques to obtain structural information (vide infra for examples). The in vivo amber suppression NNAAM method with the oocyte cell is outlined in scheme 3.8A (60, 61). The cell is CO-injectedwith two mutated RNA species: 1) the mRNA frorn the gene of interest containing an amber stop codon at the amino acid position to be mutagenized and 2) the aminoacyled amber suppressor tRNA with the unnatural aminoacyl side chain and the anti-amber antiwdon sequence (CUA). During translation, the unnatural amino acid is specifically incorporated at the appropriate position in the growing polypeptide chain encoded by the mRNA. Conventional site-directed mutagenesis was employed to introduce the arnber codon into the gene. The corresponding mRNA template was synthesized by a run-off transcription reaction with T7 RNA polymerase and the mutated gene. Synthesis of the aminoacyl amber suppressor tRNA was carried out using the three-stage protocol described in section 3.1 -2.4 (scheme 3.6). The suppressor tRNA shown in scheme 3.8B is derived from yeast tRNA Phe -in which several base changes have been made to minimize reacylation of the deacylated tRNA and maximize the suppression efficiency. A suppressor tRNA derived from eukaryotic Tefrahymena thermophila (T. thermophila) ~RNA~'"has also been employed. 6 c,A, C A Q-C * Changed to suppress *A-"* reacylation a-c O -. U A-U U-A U-A U U ~ACAC' A A "=AcUco IIIII 0 CUOUO c IlII u O OAOC C u QAA O A Q Q * C-Q C-O A-U Xenopus oocyte ---c.Measure + Changed to increase O-C suppressh effiiency A-u c A Scheme 3.8 (A) Strategy for unnatural amino acid incorporation into membrane-bound proteins in intact Xenopus laevis oocytes. The unnatural amino acid (L-configuration) side chain is denoted as R'. (B) Structure of the nonsense suppressor tRNA (derived from yeast tRNAPhe)designed to maxirnize suppression efficiency and minimize reacylation by endogenous oocyte aminoacyl-tRNA synthetases. (Diagrams obtained Nowak et al. paper (60)). Several investigators have studied the interaction between the nicotinic acetylcholine receptor (AChR) and several different ligands using unnatural amino acids incorporated at receptor binding sites (60, 62). These non-standard arnino acids are Iisted in figure 3.5A- Turcatti and collaborators reported the incorporation of 3-N-(7-nitrobenz-2- oxa-1,3-diazol4-y1)-2,34iarninopropionic acid (NBD-Dap), a fluorescent non- natural amino acid, into the G protein-coupled receptor neurokinin-2 (NK2) (figure 3.5B) (63, 64). A fIuorescence-based approach was then employed to study ligand-receptor interactions. Of particular interest is that the researchers were able to determine intermolecular distances by measuring the fluorescence resonance energy transfer between the fluorescent non-natural amino acid and a fluorescently-labelled NK2 heptapeptide antagonist. The fluorophore tethered to the ligand was tetramethylrhodamine (TMR). This was the first report of incorporation of a fluorescent unnatural amino acid into a membrane-bound protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimentai distances between a G protein-coupled receptor and its ligand by FRET. The method presented by Turcatti and colleagues can be generally applied to analysis of spatial relationships in integral membrane proteins. In a 1997 publication, Gallivan and collaborators described the incorporation of the biotin-containing amino acid biocytin (figure 3.5A) into the nicotinic acetylcholine receptor (65). Binding of 'rjl-labelled streptavidin to biotinylated receptors was used to determine highly surface-exposed residues. Figure 3.5. Unnatural amino acids (L-configuration) incorporated into the nicotinic acetylcholine receptor (AchR) binding site (A) and neurokinin-2 (NU) receptor (B) in Xenopus laevis oocytes. (C) Other unnaturat amino acids successfully introduced into membrane-bound proteins in Xenopus oocytes. The fluorescent residue is labelled. Other non-natural amino acids that have been incorporated into proteins via the arnber suppression-oocyte strategy is shown in figure 3.5C (61, 66). 3.1.3.2 In vivo incorporation with an orthogonal tRNAlaminoacyl-tRNA synthetase pair in E. coli- The aforementioned in vitro and semi-in vitro techniques for protein labelling suffer from the drawback that the labour and costs of materials are high. These considerations have motivated efforts to develop a general approach for site-specific incorporation of non-standard amino acids into proteins in vivo directly from unnatural amino acids in the growth medium. The development of techniques for in vivo NNAAM in E. coli using unnatural amino acids from the growth medium is underway (67-72).Progress was recently reported by Liu and collaborators (71). The strategy is divided into three parts (71). The first stage involves generation of an orthogonal suppressor tRNA that is not a substrate for any endogenous E. coli arninoacyl-tRNA synthetases yet functions with the prokaryotic ribosomal machinery. In the second step, a mutant aaRS is evolved that is able to aminoacylate the O-tRNA with any natural amino acid. At the same time, the synthetase must not be able to aminoacylate any endogenous tRNAs. Evolution of a mutant aaRS that charges the O-tRNA with an unnatural amino acid is the third and final part. The first two steps of the three-stage strategy has been accomplished. Two different orthogonal tRNAlsynthetase pairs have been developed. One pair was derived frorn Saccharomyces cerevisiae (S. cerevisiae) ~RNA~'"'and GlnRS (glutaminyl-tRNA synthetase) (71); while the second pair is based on the archaebacterium Methanococcus jannaschii (M. jannachio ~RNA~~and TyrRS (tyrosy 1-tRNA synthetase) (72). 3.1.4 Adaptability of non-natural amino acids to the ribosome in E. coli and rabbit reticulocyte Success of non-natural amino acid mutagenesis (in vitro or in vivo), in general, depends on acceptance of the non-standard amino acids by the E- coi;, rabbit reticulocyte, or other protein biosynthesizing machineries. It would be useful if one could predict what type of residue will be successfully incorporated and what type will be rejected during translation. The first step towards this goal requires experirnents in which a library of non-natural amino acids are tested and compared for incorporation efficiency. 3.1 A.1 Investigations using puromycin analogues Puromycin (figure 3.6A) is an inhibitor of protein biosynthesis. Due to the similarity in structure between the molecule and the 3' terminal aminoacyl adenosine residue of aminoacyl-tRNA, the inhibitor binds to the ribosomal A site and prevents the entry of the coded aa-tRNA. Like the aa-tRNA, puromycin serves as an acceptor (via the a-amino group of the p-methoxyphenylalanyl moiety) of the nascent polypeptidyl chain attached to the P-site tRNA. After peptide bond formation, the new peptidyl chain (which consists of the old peptide + the aminoacyl residue from puromycin) is attached to the ribosyl group of puromycin by an amide bond and therefore can not serve as a donor to the next aa-tRNA; as a result, chain elongation terminates (73-76). Figure 3.6. (A) Structural formulae for unmodified puromycin. (B) Puromycin analogues in which the p-methoxyphenylalanyl group is replaced with S-alkyl- and S-aryl-cysteinyl moieties. Nathans and Neidle were the first to observe that the nature of the amino acid of puromycin plays an important role in the ability of the motecule to inhibit protein synthesis (77). They discovered that the amino acid must be of L- configuration (e.g. an L-phenylalanyl analogue was just as active as puromycin whereas the D-phenylalanyl analogue did not exhibit any activity). Analogues with aromatic amino acids (e-g. L-phenylalanine, O-butoxy-L-phenylanaline,and L-tyrosine) tended to be the most potent (although the species with L-tryptophan exhibited poor inhibition). Lastly, the L-leucyl, L-prolyl, and L-glycyl analogues were inactive. The great differences in activity of the compounds with different natural amino acids were unexpected. It was anticipated that any analogue with a natural amino acid would be equally effective since each of those compounds would resernble an aminoacyl-tRNA. The basis of the specificity was unknown; however, it was hypothesized that the variation could reflect a tighter binding of the aromatic aminoacyl derivatives to the inhibited site. The general greater inhibitory effect for puromycin analogues with aromatic side chains (similar in structure to the L-phenylalanyl, and L-tyrosyl derivatives) relative to compounds with non-aromatic side chains observed by Nathans and Neidle (77) have also been confirmed by several other reports (78- 82). Puromycin analogues in which the p-methoxyphenylalanyl group is replaced with S-alkyl- and S-aryl-cysteinyl moieties (figure 3.68 (a - i) were evaluated as substrates for the peptidyl transferase reaction by Fong and Vince (83). Compounds a - f were found to terminate chain elongation while g - i were inactive. In addition, interesting trends were observed: A parallei increase in substrate efficiency accornpanied the additional hydrophobic character of the side chain as R (figure 3-68) was extended from 4 carbons (b) to 6 carbons (d) (suggesting the existence of an hydrophobic binding pocket); however, there was a drop in efficiency when 8 carbons (e) was reached. When the last rnethyl group of compound e was replaced with an hydroxyl group to produce molecule f, the activity increased dramatically. This suggested a change in environment (from hydrophobic to hydrophiiic) of the aminoacyl binding site in the ribosome. Compounds h and i were used to evaluate the bulk tolerance of the ribosome at the aminoacyl binding site; the inability of either molecule to act as acceptors of the transpeptidation reaction suggested that the increased bulk of the R group prevented the binding of the analogues to the ribosome. The results of Fong and Vince (83) also suggested that the preference for an aromatic ring by the potent puromycin analogues observed by Nathans and Neidle (77) and other investigators may be due to a strong hydrophobic interaction between the rings and the binding site in the ribosome. This hypothesis was confirmed by a report by Ariatti and Hawtrey who showed that a puromycin analogue in which the phenyl ring is substituted by a cyclohexyl ring was an inactive inhibitor (84). Hohsaka and colleagues rneasured the inhibition efficiencies for several puromycin analogues with aromatic side chains (figure 3.7) (85). Atl except 2- pyrenylalanine (compound 5) were active substrates. They also observed that the ribosome was able to discriminate the amino acid side-chains by size (Le. Figure 3.7. 1-amino acids tested for adaptability to the E. coii ribosome using puromycin analogues. The carboxyl group forms an amide bond with the 3'-amino group of puromycin (like that shown in figure 3-68). small groups generally tended to inhibit more strongly) as well as by geometry. They proposed a rnodel for the types of structures of aromatic side chains which more likely to be accepted by the ribosomal A site (figure 3.8) (85). The similarity in structure, ribosome binding site, and peptide accepting activity between puromycin and aa-tRNA suggests that correlations can be made between the adaptability of the puromycin analogues and the adapatability of aminoacyl-tRNAs with the same aminoacyl side chains. 3.1 A.2 Library screening by frame-shift suppression In 1999, Hohsaka and collaborators reported NNAAM frame-shift suppression experiments aimed at determining the incorporation efficiencies of several non-natural arnino acids with large aromatic side groups in an E. coli S30 extract as well as a rabbit reticulocyte lysate coupled transcription/translation system (57). Streptavidin was the target protein. Figure 3.9 shows a list of the aromatic residues which were tested. The investigators' goal was to explore the relationship between amino acid structure and incorporation effciency. From the E. coli S30 extract coupledftranslation experiments, it was observed that aromatic unnatural amino acid side chains were inwrporated with efficiency that depended on their shape. Aromatic groups such as 2- naphthylalanine (compound 2),p-biphenylalanine (compound 3), 2-anthïylalanine (compound 4), p-benzoylphenylaline (compound 17), and p- phenylazophenylalanine (compound 16) (figure 3.9) were incorporated efkiently. On the other hand, groups such as 9-anthrylalanine (compound 6), 9- phenanthrylalaine (compound 6).and 9-carbazoylalanine (compound 12) (figure P-S ITE A-SITE Figure 3.8. Model for predicting adaptability of aromatic non-natural amino acids (L-configuration) to the ribosornal A-site, as determined from E, coli cell-free protein synthesis inhibition experiments using 3'-N-aminoacyl analogues of puromycin. Non-natural amino acids (of the type shown in figure 3.7) with benzene rings in regions rnarked B, E, G, H, and I can adapt whereas those carrying benzene rings in the other regions are not tolerated. (Picture obtained from Hohsaka et al. PaP(85)). 3.9) were strongly rejected- The efficiencies of incorporation for the different non-standard amino acids were in fine with the efficiencies of protein synthesis inhibition by the aromatic purornycin analogues (vide ultra: section 3.1 -4.1). From the incorporation efficiency data, Hohsaka and co-workers suggested a hypothesis for allowed and excluded regions for a benzene ring in L-arylalanine- type arnino acids for acceptance by the E. coli ribosome (figure 3.1 0) (57). The pattern of incorporation efficiencies for the large aromatic non-natural amino acid residues in a rabbit reticulocyte lysate protein-synthesizing system were virtually the same as that observed in the E. coli system. The parallel amino acid dependence in the two systems suggested that the ribosome of the rabbit reticulocyte recognizes the non-standard amino acids in the same rnanner as in E- cok The only difference observed was that residues with large side groups such as pyrene, dinitrobenzene, and anthraquinone were incorporated more eficiently during translation in the rabbit reticulysate lysate than when the E coli transcription/translation system was employed. Thus suggested that the RRL system would be more appropriate for synthesizing proteins containing unnatural amino acids with large aromatic side chains. The results from these experiments together with the puromycin analogues experiments suggest that the incorporation efficiency of a non-natural amino acid may be deterrnined by, among other factors, the binding of the unnatural side chain to the ribosomal A site. Figure 3.10. Hypothesis suggested by Hohsaka et al. of allowed and forbidden regions for a benzene ring in L-arylalanine-type amino acids (see figure 3.9 for examples) for acceptance by the E. coli and rabbit reticulocyte ribosomes. Benzene rings in region A: allowed; region B: may be allowed; and X: rejected. (Diagram obtained from Hohsaka et al. publication (57)). 3.1.5 A novel method for site-specific labelling of proteins with non-natural reporter groups (t~~~~~~-mediated protein labelling) We wished to develop a novel methodology for site-specific protein labelling that incurporated the best features of non-natural arnino acid mutagenesis with the ease of post-translational chemical labelling. An outline of the proposed technique which we will refer to as tR~~'~-mediatednon-natural amino acid mutagenesis (or tR~A~~~-mediatedprotein labelling) is shown in scheme 3.9. The first step involves enzymatic synthesis of a C~S-~RNA~" construct from E. coIi ~RNA'~,1-cysteine, E. coli cysteinyl-tRNA synthetase, and ATP. The aminoacylated tRNA is then chemically rnodified with a thiol-reactive labelling reagent. In the final stage, the chemically-modified C~S-~RNA~~~ construct is combined with a plasmid carrying the gene of interest in a rabbit reticulocyte lysate coupled transcription/translation system that is capable of transcription and protein synthesis. During translation, the UGC and UGU codons in the mRNA, which specify for unmodified cysteine under normal circumstances, are translated as the chemically-modified arnino acid. ~RNA'~'- mediated NNAAM is essentially an extension of the methodology that was originally employed by Chapeville and CO-workersfor the introduction of alanine and cysteic acid into the poly (UG)-encoded polypeptide and globin chains (vide ultra: section 3.1 -2.1). Unlike those investigators, however, we wish to modify the cysteinyl side-chain with alkylating agents and incorporate amino acids with more complex side chains. Thiol-specific Aminoacylation labelling reagent w c > GCA L-cysteine GCA ln vitro transcription ACG Plasmid - 5' UG ClU 3' mRNA / In vitro traan,atiin Mutant enzyme with unnatural amino acid site-specifically incorporated Scheme 3.9. Outline of a novel non-natural amino acid mutagenesis technique based on the use of chemically-modified Cys-tRNACYS(tRNACYs-mediated non-natural amino acid mutagenesis). t~~A'~-mediatednon-natural amino acid mutagenesis is, overall, sirnilar to the technique that utilizes chernically-modified I~S~I-~RNA~~~.In both cases, the introduction of a non-standard amino acid relies on in vitro chernical modification of the side chah of a natural amino acid that has been enzymatically arninoacylated to the cognate tRNA. Compared to lysine, however, cysteine occurs less frequently in proteins. On average, lysine represents 6.6% of a protein's amino acid content (86). Cysteine, on the other hand, is typically in the 1.7% range (5). For this reason, protein labelling at multiple sites is less of a problem from our proposed technique than the established method. In principle, t~NA'Krnediated protein labelling has two main advantages over conventional chernical modification of protein cysteine residues. It is highly site-specific and not restricted to positions which are surface-exposed. The use of chernically-modified C~S-~RNA'~is more flexible than the employment of N-acylaminoacyi-tRNA derivatives. While the former enables insertion of unnatural amino acids anywhere along a protein sequence, the latter restricts protein labelling to the N-terminus. t~N~'~~-mediatedNNAAM is not as versatile as the arnber and frame-shift suppression techniques. While the former still has to contend with multiple-site labelling, the latter techniques allow site-specific introduction of a single non- natural residue. This problem could be circumvented by employment of conventional site-directed mutagensis to rernove undesired excess cysteine residues. Though removal of several critical cysteine arnino acids may create problems to protein stability or function, the risk is less with cysteine than with lysine (as there are generally more lysines than cysteines to mutate). Furthermore, a cysteine-specific tRNA (of E. coli origin) in which the anticodon has been mutated to UCA (Le. a triplet that is cornplementary to the UGA (opal) termination codon (or anti-opal)) from GCA is available in our laboratory. We have observed that the mutant tRNA can be aminoacylated with L-cysteine using E. coli cysteinyl-tRNA synthetase (the experimental details for which is not discussed here), albeit with lower efficiency (-5 - 8-fold (measured by Dr. Ananda Seneviratne)) than in the situation with the native tRNA (the GCA anticodon is one of the identity deterrninants of E. coli ~RNA'"). As for Cys- ~RNA'~,C~S-~RNA"~~ can be chemically modified with a thiol-reactive compound. When this labelled tRNA and a plasrnid containing a gene with a premature opal codon are combined in an in vitro coupled transcriptionftranslation system, the chemically-modified cysteine from the synthetic aa-tRNA should be incorporated into the target protein by suppression of the opal codon during translation (assuming that the label is tolerated by the protein synthesis system). This procedure is outlined in scheme 3.1 0. In this manner, the unnatural residue is introduced at only one position and the opal codon can be placed anywhere in the gene. The use of aminoacyl opal suppressor tRNAs is analogous to the employment of aminoacyl frame-shift and amber suppressor tRNAs. However, in Our situation, chemical aminoacylation is not necessary since the anti-opal tRNA can be aminoacylated using CysRS, unlike the anti-amber and frame-shift suppressor tRNA which are not substrates for any aminoacyl-tRNA synthetase. Thus, the synthesis of chemically-modified C~S-~RNA"~~is less time-consuming, less costly, and less labour-intensive than the preparation of the other aminoacyl suppressor tRNAs. From this point of view, NNAAM via the use of t~~~~verivedaminoacyl opal suppressor tRNAs is more powerful than amber and frame-shift suppression NNAAM. We chose to test the t~~~~~'-rnediatedprocedure rather than the t~~~"'~-mediatedmethod because the synthesis of chemically-modified cys-t~~ACFappeared to be simpler than the preparation of labelled C~S-~RNA"~~.Work on testing the latter procedure is currently under way in our laboratory (Dr. Ananda Seneviratne) (not discussed here). The use of chemically-modified C~S-~RNA'"constructs would expand the scope of techniques for ribosome-based protein labeling. Furthermore, our proposed technique could be used in conjunction with a different method such that two or more different probes can be concurrently introduced into the same protein. In our initial experiments towards developing a t~~~~~'-mediated methodology for incorporating complex non-standard amino acids, we atternpted to use IAF-labelled C~S-~RNA~"and BIADD-labelled C~S-~RNA~~~(structures in chapter I(figures 1.16a and d, respectively)) to incorporate a biotin derivative and fluorescein, respectively. These labels were chosen for several reasons: 1) They can be detected at low concentrations (the molar absorptivity and quantum yield of Ruorescein are 75 000 ~-'crn-'and 0.71, respectively); 2) They are soluble in DMSO-water solution with c 47% DMSO, which was necessary to avoid labeiling of the tRNA backbone (chapter 1: section 1.4.6); and 3) They are cornmercially available in a thiol-reactive for- Our choice for a test protein was luciferase from the firefiy Photinus pyralis (P. pyraiis). Luciferase is an enzyme which uses ATP, molecular oxygen, and the heterocyclic cornpound Iuciferin to generate visible light (87). It is a small (MW = 63 000) and water-soluble protein. lt has been shown by Kolb and colleagues that luciferase protein that is synthesized in a cell-free systern folds into a native structure and exhibits full activity (88).Additionally, the enzyme does not require post-translational processing or modification for activity. Its high-resolution three dimensional structure has been reported (89). Finally, its activity could be detected by a simple and sensitive assay. An aliquot of the translation protein is combined with luciferase assay reagent (Promega) and the intensity of the emitted light is measured using a luminometer. The protein contains 4 cysteine residues (89). In a recent publication, Kumita and co- workers showed that these can be mutated to serine and the enzyme retains bioiuminescence activity (90). 3.2 Materials Plasmids N2R, N3R, N4R, N5R, and NCFll (with SP6 promotors) encoding CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) fragments were gifts given by Mingang Chen (University of Texas, Galveston). Plasmids SNCF12 and SNCF56 (with T7 promotors) encoding CFTR fragments, were gifts provided by Natalie Goto (University of Toronto). Plasmid pBQ6.2 (with a T7 promotor) encoding full-length CFTR was a gift from Dr. J. Rommens (University of Toronto). [35~]-methionineand fluorographic reagent for treatment of polyacrylamide gels (Amplify) were purchased from Amersham Life Science (Baie dlUrfe, Quebec). Broad range protein molecular weight markers and biotinylated molecular weight markers were purchased from BIO-RAD (Mississauga, ON). TNT@rabbit reticulocyte lysate coupled transcription/translation kits, nas sin@ ribonuclease inhibitor, luciferase assay reagent, and a plasrnid containing the gene for P. pyralis luciferase (pPOLY(A)- luc (SP6)) were purchased from Promega (Madison, WI). The acquisition of al1 other materials was described in chapters 1 (section 1.2) and 2 (section 2.2). 3.3 Experimental Procedures 3.3.1 Partial purification of E. coli cysteinyl-tRNA synthetase from programmed JMlO1 cells and preparation of denatured CysRS E coii cysteinyl-tRNA synthetase (active and denatured) was prepared and stored in the same manner as described in chapter 1 (section 1.3.1 ). 3.3.2 Aminoacylation of ~RNA'~'with L-cysteine ~~stein~1-t~~~~~was synthesized and stored using the same protocols described in chapter 1 (section 1.3.5). For synthesis of [35~]-~ys-t~~~CF,30 pL of [3s~]-cysteinewith a specific activity of 1000 Cilmmol from a freshly prepared stock was included in the 50 pi aminoacylation reaction. The samples were either stored in a -80 OCfreezer or used directly in the chemicai modification or RRL coupled transcription/translation experirnents. 3.3.3 Synthesis and purification of BIADD- and IAF-labelled cystei nyl-t~~~CYs The cysteinyl side-chain in non-radiolabelled cy~tein~l-t~~~~~~was chemically rnodified with 5'-iodoacetamidofluorescein (IAF) and (+)-biotinyl- iodoacetamidyl-3,6-dioxaoctanediamine (BIADD) (chapter 1: figures 1.16a and d, respectively) in the sarne manner as described in chapter 1 (section 1.3.1 0, scherne 1.2) and chapter 2 (section 2.3.11, respectively. The adducts were purified by reverse-phase high performance liquid chromatography using a C8 column and stored following the same protocols described in chapter 2 (section 2.3.3). The purification step was necessary because the unaminoacylated ~RNA'" and unlabelled C~S-~RNA'"(arisen from the incomplete aminoacylation and alkylation reactions (described in chapfer 1 (sections 1.4.1 and i -4.6, respectively), respectively) could compete with the modified tRNAs during the protein synthesis reactions. IAF- and BlAD D-labelled C~S-~RNA'~were either stored in a -80°C freezer or used directly in the protein synthesis experiments. 3.3.4 TNPrabbit reticulocyte coupled transcriptionltranslation reactions TNT@rrabbit reticulocyte lysate coupled transcription/translation reactions were assembled on ice with a total volume of 25 yL in each 1.5 mL microcentrifuge tube. For reactions that were not supplemented with an exogenously formed aminoacyl-tRNA construct, the lysate was added to the tube last. For reactions that were supplemented with an exogenously formed aminoacyl-tRNA construct, the lysate and tRNA construct were added second last and last, respectively. Al1 the other cornponents were assembled in an arbitrary order. Gloves were worn at al1 times to avoid ribonuclease contamination. Components of the TNT@kit as well as franscendTMtRNA were handled and stored according to the manufacturer's instructions (Promega technical bulletins TB126 and TB182). Except for the luciferase plasmid which waç purchased, al1 plasmid constructs were prepared by the standard alkaline lysis rnethod described by Sambrook et al. (91). Al 1 plasmid constructs were stored at -80 OC in TE (Tris-EDTA) buffer (10 mM Tris, pH 8.0 (HCI), 1 mM EDTA) at a concentration of approximately 0.5 pg/pL. Stocks of radiolabelled amino acids were stored at -80 OC to avoid oxidation. They were minimally exposed to the atmosphere and ambient temperatures when aliquots were required. 3.3.4.1 Protein synthesis reaction programmed with plasmid for luciferase or a CFTR fragment and supplemented with [35~]-methionine A typical25 pL reaction was assembled by combining 12.5 pL of TNT@ lysate, 1 pL of TNT@reaction buffer (components not specified by the manufacturer), 0.5 pL of TNT@ SPGTn RNA polymerase (concentration and activity as determined by the supplier (not specified)), 0.5 pL of amino acid mixture minus methionine (1 mM), 0.5 pl of me as in" ribonuclease inhibitor (40 unitslpL (units of activity not specified)), -1 pg plasmid DNA (pPOLY(A)-luc (SPG), SNCFl2, SNCF56, N2R1N3R, N4R, N5R1NCFII, or pBQ6.2), 0.3 pL of Triton X-100 (only for experiments with plasmids SNCF12, SNCF56, and pBQ6-2). 2 pL of [3s~]-methionine(1 000 Cilmmol, 10 pCiIpL), and the required amount of nuclease-free H20. A reaction in which no DNA was included was used as a control to allow measurement of any background incorporation of radiolabelled amino acids. The reaction was incubated in a 30 OC regulated water bath for -90 min. A 1 pL aliquot was used for analysis by SDS-PAGE on a 12.5% polyacrylamide gel in combination with autoradiography to detect transiation products while a 2 pL aliquot was used to determine product yield (vide infra: section 3-3.6).For the transcriptionltranslation with luciferase, a separate 5 pL aliquot was used in a photographic assay to detect luminescence. The remaining transcription/translation mixture, after the amount required for analysis had been removed, was stored in a -20 OC freezer. 3.3.4.2 Protein synthesis reaction programmed with plasmid for luciferase and supplemented with either C5s]-cysteine or exogenously formed PSI-C~S-~RNA~~~ A typical 25 pL reaction was assembled by combining 12.5 pL O~TNP lysate, 1 pL of TNT@reaction buffer (components not specified by the manufacturer), 0.5 pL of TNPSP6 RNA polymerase (concentration and activity as deterrnined by the supplier (not specified)), 0.5 pL of amino acid mixture minus cysteine (1 mM), 0.5 pL of a as in" ribonuclease inhibitor (40 units/jL (units of activity not specified)), -1 pg of plasmid DNA (pPOLY(A)-luc (SPG)), [35~]-cysteineor exogenously formed [35~]-~ys-t~~~CF(the amount added (in terms of specific activity and concentration) is specified in the relevant figure legends), and the required amount of nuclease-free H20. The reaction was incubated in a 30 OCregulated water bath for -90 min. Translation products were detected by 12.5% SDS-PAGE in combination with autoradiography. Five or fifteen microlitre of the sample was analyzed (the exact arnount is indicated in the relevant figure legends). The remaining transcription/translation mixture was stored at -20 OC. 3.3.4.3 Protein synthesis reaction programmed with plasmid for for luciferase and supplemented with BIADD-labelled C~S-~RNA~~*,IAF-labelled C~S-~RNA~~~,or ~ranscend~~ tRNA A typicai 25 FL reaction was assembled by combining 12.5 pL of TNT@ lysate, 1 pL of TNPreaction buffer (components not specified), 0.5 pL of TNP SP6 RNA polymerase (concentration and activity as determined by the supplier (not specified)), 0.5 pL of amino acid mixture minus methionine (1 mM), 0.5 pL of asin in@ ribonuclease inhibitor (40 unitslpl (units of activity not specified)), -1 pg of plasmid DNA (pPOLY(A)-luc (SP6)), 2 pL of [35~]-methionine(1000 CVmmol, 10 pCi/pL), a rnodified tRNA construct (BIADD-labelled C~S-~RNA~~~,IAF- labelled C~S-~RNA~~~,or ~ranscend~~ tRNA (biotinylated lysyl-tF3NALYS (Promega))) (the amount added is indicated in the relevant figure legends), and the required amount of nuclease-free H20. The reaction was incubated in a 30 OCregulated water bath for -90 min. Translation products were detected by 12.5% SDS-PAGE in combination with autoradiography as well as either western blot (for protein synthesis reactions with B IADD-labelled C~S-~RNA~~or ~ranscend~~tRNA) or fluorescence imaging (for protein synthesis reactions with IAF-labelled C~S-~RNA~").From the experiments with ~ranscend~~tRNA and BIADD-labelled C~S-~RNA~~,a 5 pL aliquot of the transcription/translation reaction was analyzed. For the experiment with IAF-labelled C~S-~RNA~~,a 12 PL aliquot was analyzed. The remaining transcription/translation mixture was stored in a -20 OCfreezer. 3.3.5 Analysis of translation products 3.3.5.q Autoradiography (using autoradiographic film) After SDS-PAGE on a 12.5% polyacrylamide gel, staining and fixing with a solution made up of O. 1% Coomassie blue R-250, 40% methanol, and 10% acetic acid, and destaining with a solution containing 40% methanol and 10% acetic acid, the gel was treated with a Ruorographic reagent (AmpliQ) for -1 5 min. The gel was dried on a piece of ~hatman@filter paper using a gel drier. The dried gel was exposed on a sheet of high performance autoradiographic film (~yperfïlm~~MP (Amersham Life Science)) in a cassette in a -80 OC freezer. The duration of exposure ranged from 2 to 72 h and depended on the radioactivity of the samples. Typically, a gel was exposed until bands were observed and background was not too high- The film was developed using a Kodak X-Omat 2000A processor. 3.3.5.2 Autoradiography (using a phosphorimager) Alternatively, the dried gel was exposed on a phosphor screen in a cassette at room temperature for -2h. Two hours tater, the bands were detected by phosphorirnaging (described in chapter 1 (section 1.3.7). 3.3.5.3 Fluorescence irnaging After SDS-PAGE on a 12.5% polyacrylamide gel, the unstained gel was placed directly in a phosphorimager. The gel was scanned using the blue fluorescence setting and a scan voltage of 900 V. Bands were monitored by fluorescence imaging on a rnini UV transilluminator as well. 3.3.5.4 Western blot Western blots were preformed using a Mini ~rans- lot@ electrophoretic transfer cell (BIO-RAD). After SDS-PAGE on a 12.5 % polyacrylamide gel, the unstained gel was rinsed with transfer buffer (25 mM Tris and 192 mM glycine) for -1 5 min on a shaker sitting at room temperature. The blot "sandwich" was ôssembled according to BIO-RAD'S instruction manual. The type of membrane used was polyvinylidene difluoride (PVDF) (BIO-RAD). The membrane was presoaked in methanol for -5 min prior to use. The electorphoretic transfer assembly was assembled according to the manufacturer's instructions. A stirring bar was included in the assembly to ensure uniform distribution of heat and buffer ions during the run. The electrophoretic transfer was perforrned with the transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3 (no acid added)) at room temperature at constant voltage (30V) and current (90 mA) overnight. After the electrophoresis was complete, the membrane was rinsed with -1 5 mL of TBS (20 rnM Tris, pH 7.5 (HCI), 150 mM NaCI) at room temperature on a shaker for -5 min. During this as well as subsequent rinses, the membrane was placed in a shallow vesse1 (with similar dimensions as the membrane) with the protein side facing up and the membrane was prevented from drying out. Fifteen minutes later, the TBS was removed and replaced with -1 5 mL of TBST (TBS + 0.5% ~ween"20). The membrane was rinsed in the new solution for -1 h. The purpose of this step was to use the ~ween"20 molecules to block al1 unoccupied sites on the membrane. In this way, there should be less background from the colorimetric assay to be performed later (vide infra). After i h, the TBST solution was discarded, To the membrane was added a fresh 15 mL aliquot of TBST with alkaline phosphatase-conjugated streptavidin (3 pg/mL). The membrane was rinsed in this solution or -1 h. In this step, the alkaline-phosphatase conjugated streptavidin molecules should bind to any membrane-bound proteins containing an exposed biotin tag. One hour later, the solution was discarded. The membrane was then rinsed twice with two separate aliquots of 15 mL of TBST (-1 min rinse each) and twice with two separate aliquots of 15 mL of TBS (-1 min rinse each). Ten millilitre of freshly prepared alkaline phosphatase conjugate substrate solution (NBT/BCIP), prepared according to the manufacturer's (BIO- RAD) instructions, was added to the membrane. The membrane was rinsed in the substrate sotution until bands were visible (-15 min) (for chemistry see chapter 2 (scheme 2.1 A)). Biotin-containing bands appeared purple. After bands were detected at a reasonable intensity, the substrate solution was discarded and the membrane was rinsed twice with two separate aliquots of -1 5 mL of H20 (-5 min rinse each). The membrane was air-dried (overnight), taped ont0 a piece of whatmanmfilter paper, and then stored in a manila envelop to avoid light-induced discoloration of the bands. 3.3.5.5 Photographic luciferase assay Photographic luciferase assays were performed according to the instructions specified by the supplier of the luciferase assay reagent (Promega, technical bulletin TB126). 3.3.6 Determination of translation protein yield in a rabbit reticulocyte lysate coupled transcriptionltranslation reaction prograrnmed with the plasmid for luciferase or a CFTR fragment and supplemented with [35~]-methionine The following protocol was adapted from Promega technical bulletin TB126 ('Detemination of Percent Incorporation of Radioactive Label"): After the 25 pL rabbit reticulocyte lysate coupled transcription/translation reaction programmed with pPOLY(A)-luc (SP6), SNCF12, SNCF56, N2R, N3R, N4R, N5R, or NCFll and supplernented with [3s~]-methioninewas complete, 2 pl was removed and added to 98 pl of a solution consisting of 1M NaOH and 2% Hz02 in a 1.5 mL microcentrifuge tube. The tube was vortexed for -30 s and then incubated in a 37 OC regulated water bath for 10 min. At the end of the incubation, 900 pL of an ice-cold solution containing 50% trichloroacetic acid (TCA) and 4% tryptone was added to the tube to precipitate the translation product. The mixture was placed on ice for -30 min. Two hundred fifty microiitre of the mixture was then applied to a glass fiber filter circle (G6) (Fisher Scientific) (diameter: 2.4 cm) which had been pre-wetted with 1 mL of a cold solution made up of 5% TCA. The precipitated translation products were collected by vacuum filtration with an aspirator. The filter was rinsed with 1 mL of the ice-cold 5% TCA solution three times. It was then rinsed with 1 mL of acetone. The filter was dried under a heat lamp for 10 min. A total of three 250 pL aliquots from the original precipitated mixture were filtered to check for reproducibility. The filters were each placed in -2 rnL of liquid scintillation counting cocktail (cytoscintTM) in a 20 mL glass vial. The vials were counted using a liquid scintillation counter. From the counts, the amount of protein produced in a 50 pL coupled transcription/translation reaction was calculated since the number of methionine residues was known. 3.4 Results and Discussion We wished to demonstrate that chemically-modified cysteinyl-~RNA'" could be used to deliver unnatural amino acids into proteins (scheme 3.9). The process occurs in three stages: First, E. coi ~RNA'" is enzymatically charged with cysteine. The cysteinyl side chain of C~S-~RNA~~~is alkylated with a thiol- reactive compound, The modified tRNA constnict is then combined with a plasmid containing the gene of interest (with one or more codons for cysteine) in an in vitro rabbit reticulocyte lysate coupled transcription/translation reaction. We attempted to use semi-synthetic tRNA constructs IAF-labelled C~S-~RNA~"and BIADD-labelled ~ys-~RNA'" (chapter 1: figures 1.1 6a and dl respectively) to introduce the fluorophore Ruorescein and a biotin derivative, respectively, into native firefly luciferase. 3.4.1 Protein yields from rabbit reticulocyte lysate coupled transcriptionltranslation reactions programmed with the genes for luciferase and CFTR fragments and supplemented with [35~]-methionine The cell-free protein biosynthesis system employed for our experiments was rabbit reticulocyte lysate. We chose this system for two main reasons. First, it was commercially available. Second, its origin was different from that of the modified cysteine-specific tRNA. A heterologous expression system was mandatory to minimize.enzyrnatic deacylation of chemically-modified Cys- ~RNA'" and aminoacylation of ~RNA'~'(after deacylation of the labelled tRNA) with cysteine by endogenous cysteinyl-tRNA synthetases. In an effort to determine the efficiency of protein synthesis in the rabbit reticulocyte lysate system, as prepared by Promega, we performed an experiment to determine the amount of protein produced in a 50 pL coupled transcription/translation reaction that was programmed with the plasmid construct for luciferase and supplemented with [35~]-methionine.The result is shown in table 3.7. The yield obtained was 16 ng (or 256 fmol) per 50 pl reaction. For cornparison purposes, the yields for the syntheses of CFTR (Cystic Fibross Transmembrane Conductance Regulator) fragments were deterrnined as well (table 3.1). CFTR is a complex integral membrane protein (MW = 168 000). It is a regulated chloride-selective channel which is defective in people with the cystic fibrosis (CF) disorder. Development of anti-CF drugs requires knowledge about the three-dimensional structure of the protein. For this reason, CFTR is a particularly attractive application for non-natural amino acid mutagenesis. SNCF12 and N2R denote fragments which contain the first two transmembrane (TM) segments; SNCF56 denotes a fragment which contains the fifth and sixth TM segments; N3R, N4R1and N5R denote fragments which comprise the first three, four, and five, respectively, TM segments; finally, NCFll and pBQ6.2 denote the first protein half and whole protein, respectively. Synthesis of each of the proteins was detected by SDS-PAGE in conjunction with autoradiography (figure 3.1 IA). For luciferase, bioluminescence activity was detected with a photographic assay (figure 3.1 18). A general trend observed in table 3.1 is that the yield increases as the size of the synthesized protein decreases. For example, while the small CFTR fragments SNCF12, SNCF56, N2R, N3R, N4R1 cc-OS% 42 Cz ab c d e fgh i j k 1 Figure 3.1 1. (A) Autoradiographic detection of radiolabelled proteins synthesized in 25 pL rabbit reticulocyte lysate coupled transcriptionltranslation reactions supplemented with [35S]-methionineand programmed with plasmid N2R (lane a), N3R (lane b), N4R (lane c), N5R (lane d), NCFll (lane e), pPOLY(A)-luc (SP6) (lane f), SNCF56 (lane j), and SNCF12 (lane k). No radiolabelled protein was detected when the pBQ6.2 plasmid (which encodes full-length CFTR) (lane i) or no plasmid (lane g) was included in the coupled transcriptionltranslation reaction. Lanes h and I contain protein molecular weight markers. From each reaction, a 1 pL aliquot was analyzed. (B) Luciferase activity (lane a), as detected with a photographic assay. No activity was observed when pPOLY(A)-luc (SP6) was not included in the protein synthesis reaction (lane b). Aliquots of 5 pL from the reaction mixtures were assayed. N5R, and NCFII (MW = 26 000,25000, 53 000,59 000,69 000,72 000, and 63 000, respectively) were detected (1 - 11 ng or 9 - 178 fmol per 50 pL reaction), synthesis of full-length CFTR (MW = 486 000) was ver'weak (table 3.1 and figure 3.1 IA). Another general trend that is evident is that the amount of product increases as the water-solubility of the protein increases. For instance, synthesis of luciferase (a water-soluble protein) (256 fmol) was more efficient than production of the CFTR fragments (less water-soluble) (9 - 178 fmol) (see figure 3.1 1A). IVa radiolabelled protein was detected in a transcription/translation reaction that was supplemented with radiolabelled amino acids but no plasmid (figure 3.1 1A (lane g)). We have determined that the Iower Iimit of detection for 5-iodoacetamidofluorescein is -0.03 pmo1/100 pL (in methanol) in a Perkin Elmer LS5OB spectrofluorimeter. The aforementioned protein synthesis experiments showed that the amount of protein synthesized in a rabbit reticulocyte lysate coupled transcription/translation system ranges from 0.01 (for the N4R CFTR fragment) to 0.2 (for luciferase) pmol per 100 plreaction. This means that a translation product with just one fluor incorporated should be detectable by fluorescence spectroscopy. 3.4.2 Incorporation of [35~]-~steine into luciferase using exogenously formed [3 Y~]-cysteinyl-t~~~~~' Our first attempt at using an exogenously formed aminoacyl-~RNA'~' construct to insert amino acids at positions that correspond to the UGU and UGC codons (triplets that encode cysteine under native conditions) involved a coupled transcription/translation reaction that was programrned with the plasmid for luciferase and supplemented with exogenously formed [35~]-cysteinyl-t~~~cF. What we expected to observe was introduction of radiolabelled cysteine into 1uciferase via the radiotabelled aa-tRNA. We performed a series of pPOLY(A)-luc (SP6)-programmed transcription/translation experiments in which different amounts (in terms of concentration as well as specific activity) of free [35~]-cysteinewere included. The translation products from these experiments were analyzed by 12.5% SDS- PAGE in combination with autoradiography. The results are shown in figure 3.12. The lower limit of detection was found to correspond to a source of [%]- cysteine with a concentration and specific activity of 0.1 mM and 5.42 X 1o6 dpmlnrnol, respectively (lane d). No luciferase protein was detected when no plasmid was included in the transcription/translation reaction. . A transcriptiodtranslation reaction supplernented with exogenously formed [35~]-cysteinyl-t~~~CF(obtained from a stock with a concentration and specific activity of 18 pM and 6.66 X 1o6 dpmlnrnol, respectively) was then performed. Figure 3.1 3 (lane c) shows the autoradiographic results obtained. A reaction in which the [35~]-~ys-t~~~CFconstruct was substituted by free [35~]- cysteine (obtained from a stock with a concentration and specific activity of 45 pM and 6.66 X 1o6 dpmlnmol, respectively) was carried out as well (figure 3.1 3 (Iane a)). A band corresponding to radiolabelled luciferase was detected in each experiment. No luciferase protein was detected when no plasmid was included in the transcription/translation reaction that was supplemented with the hot cysteine (Iane b). From the autoradiographic results shown in figure 3.1 3, it was radioiabet led luciferase Figure 3.A 2. Autoradiographic detection of radiolabelled proteins synthesized in 25 pL rabbit reticulocyte Iysate coupled transcription/translation reaction programmed with the plasmid for luciferase and supplernented with 2 pl of [35S]-cysteine. Concentration and specific activity, respectively, for the [35S]-cysteinestock: 0.01 mM and 2.22 X 1O9 dpmlnmol (lane a); 0.1 mM and 2.22 X 1O6 dpmlnmol (lane b); 0.1 mM and 4.22 X 1O6 dpm/nmo! (lane c); 0.1 mM and 5.42 X 1O6 dpmlnrnol (lane d); 0.1 mM and 7.12 X 1 O6 dprnlnmol (lane e); 0.1 mM and 8.62 X IO6 dpmfnmol (lane f); 0.1 mM and 1.O X 10' dpminmol (lane g). From each experiment, a 15 pL sample was analyzed. No luciferase protein was detected when no piasmid was included in the coupled transcriptionitranslation reaction. t radiolabelled luciferase [35S]-cysteine from deacylated [35S]-Cy~-tRN ACYs Figure 3.13. Autoradiographic detection of radiolabelled proteins synthesized in a 25 pL rabbit reticulocyte lysate coupled transcriptionltranslation reaction programmed with the plasmid for luciferase and supplemented with either 4 pL of free r5S]-cysteine (45 pM at 6.66 X IO6 dpmlnmol) (lane a) or 4 pL of [3SS]-Cys-tRNAC~S(18 PM at 6.66 X 106 dpmlnmol) (lane c). From each experiment, a 5 pL sample was analyzed. No luciferase protein was detected when no plasrnid was included in a transcription/translation reaction supplernented with the free hot cysteine (lane b). inconclusive whether [3s~]-cysteinewas incorporated into luciferase via the exogenously fonned radiolabelled aminoacyl-tRNA construct. It is conceivable that [35~]-cysteinecould have been incorporated via aminoacylation of the endogenous rabbit ~RNA'~or E coli ~RNA'~~(after hydrolysis of the [35~]-~ys- ~RNA'") with [35~]-cysteine(from the deacylated [35~]-~ys-t~~~Cp)by the endogenous rabbit cysteinyl-tRNA synthetase. After all, radiolabelled luciferase was detected in the transcription/translation reaction supplemented with free [3s~]-cysteineinstead of [35~]-cysteinyl-t~~~Cys~However, radiolabelled 13%]- C~S-~RNA~~still seemed to be present in the autoradiograph (figure 3.1 3 (lane c). To resolve the ambiguity, a transcriptionJtranslation experiment with chemically-modified C~S-~RNA~"was performed. Chernically-modified cysteine that has hydrolyzed from intact arninoacyl-tRNA can not be re-attached to ~RNA~"(rabbit or E. col0 by endogenous rabbit reticulocyte lysate cysteinyl- tRNA synthetase because the enzyme is highly selectively for cysteine. 3.4.3 Translation with ~ranscend~~tRNA and BIADD-labelled CYS-~RNA~Y~ We attempted to incorporate a biotin derivative into luciferase using ~ranscend~~tRNA (structure in chapter 2: figure 2.3B), which is essentially a biotinylated lysyl-t~~~~~~,and BIADD-labelled C~S-~RNA'" (chapter 1: figure 1-1 6d). The former tRNA construct was purchased from Promega. Three coupled transcriptionJtranslation reactions programmed with the plasrnid for luciferase were carried out. One reaction was supplemented with ~ranscend~~ IRNA, a second contained the less hydrophobic species of BIADD-labelled Cys- ~RNA'~'(1 8 min retention time in RP-HPLC (see chapter 2: section 2.4.2)), and the last one was supplemented with the more hydrophobic BIADD-C~S-~RNA~" species (RT = 22 min in RP-HPLC (see chapter 2: section 2.4.2)). Ail three reactions were supplemented with [35~]-rnethionine.Translation products from the reactions were analyzed by autoradiography as well as on Western blots. The results are shown in figures 3.1 4A and 6,respectively. Luciferase protein radiolabelled with [3s~]-methioninewas detected in al1 experiments (figure 3.1 4A (lanes a, c, and d)). No luciferase protein was found when no plasmid was included in the transcriptionltranslation reaction (figure 3.1 4A (lane b)). In the western blot, biotinylated luciferase was obsewed only in the reaction with ~ranscend~~tRNA (figure 3.148 (lane a)). Approximately 25 - 33% of the lysine residues in the translation products (which corresponds to nine for luciferase) are ranaomly biotinylated (Promega technical bulletin TB?82). Biotinylated luciferase was not detected in the reactions with BIADD-labelled cys-t~~ACF(figure 3.146 (lanes c and d)). The synthesis of biotin-free radiolabelled luciferase (figure 3.14A (lanes c and d)) in the presence of the biotinylated ~~stein~l-t~~~~~~constructs appears to be due to aminoacylation of ~RNA'~'(endogenous or from BIADD-C~S-~RNA~~* after deacylation) with cysteine (endogenous and from the supplemented amino acid mixture) by endogenous rabbit cysteinyl-tRNA synthetase. The absence of biotinylated luciferase does not appear to be a consequence of protein production inhibition by the synthetic tRNA construct since [35~]-labelledproteins was observed ai normal levels. .'. >' ...... <::..:*..A ...... ,,.,?..,.>, radiolabelled . . ' " '"Y ...... , . luciferase ...... ,: . I. ,.._, + ...... ,&.ru.>,, ...... ,. ,,. biotinylated.lueifera~e'';:y,.. .,., :! -...... A',' . ;,%.,388 ...... '_..: kt.&;,...... , , , , . , , %,'.' , , ..... , .* ...... , Figure 3.14. Autoradiographic detection of radiolabelled proteins (A) and western blot detection of biotinylated proteins (B) synthesized in a 25 pL rabbit reticulocyte lysate coupled transcriptionltranslation reaction programmed with the plasmid for luciferase and supplemented with 2 pL of [35S]-rnethionine(1000 Cilmmol) and 1 CLof TranscendTMtRNA (28 FM) (lane a); 2 yL of BIADD-labelled Cys-tRNACyS(species with an 18 min RT) (14 pM) (lane c); or 0.7 pL of BIADD-labelled Cys-tRNACYS(species with a 22 min RT) (41 FM) (lane d). No luciferase protein was detected when no plasmid was included in the transcriptionltranslation reaction (lane b). From each experiment, a 5 pL aliquot was analyzed. (Biotinylated protein markers: 200 000, 116 000, 97 400, 66 200,45 000,31 000,21 500, and 14 400). The sensitivity of biotin detection using streptavidin-conjugated alkaline phosphatase in combination with a colorimetric assay has been reported to be equivalent to that achieved with [35~]-rnethionineincorporation and autoradiographic detection (Promega technical bulletin TB182). This suggests that cysteine is easier to incorporate, either via rabbit or E. coli ~RNA'", than the B IAD D-Cys moiety. Poor adaptability of the biotin derivative to the ribosome would account for the difficuky of incorporation. It has been shown by several investigators that, in general, puromycin analogues with more hydrophobic side chains are better inhibitors of protein synthesis than those with groups which are less hydrophobic (see section 3.1 -4.1) (77-83), thus suggesting that the former molecules have stronger association than the latter with the ribosome. These observations are in line with the successful introduction of the biotin derivative frorn the ~ranscend~~ tRNA and rejection of the biotin group from BIADD-labelled C~S-~RNA~~~because the BIADD-C~S-~RNA~"is water-soluble (Le. more polar) while the biotinylated lysyl-tRNA is water-insolub le (Le. less polar). Poor interaction between the BIADD-cysteinyl side chain and the eiongation factor Tu (EF-Tu) would also explain why the amino acid failed to incorporate. As described in chapter 1 (section 1.1 -2.1), during translation, EF- Tu's role is to form a ternary complex with the aa-tRNA and a molecule of GTP and transfer the aa-tRNA to the ribosomal A site. ln chapter 1, it was observed that IAF-labelled, AIASS-labelled, and IAEDANS-labelled C~S-~RNA'" (which contain aminoacyl side chains that are bulkier than the BIADD-labelled cysteinyl moiety) (chapter 1: figures 1.16a1 b, c. and d) are able to form ternary complexes with EF-Tu-GTP, and molecular modelling suggested that groups which are narrow and flexible near the cysteine attachment site in chemically-modified Cys- ~RNA'~should be tolerated by the EF-TU binding site. This suggests that BIADD-labelled C~S-~RNA~"should be able to forrn ternary complexes as well because the biotin derivative is not as bulky as the fiuorophores and parts near the cysteine attachment site are narrower than those in the fluorescent aa-tRNAs (cf. figures 1-1 6a, b, c, and d). However, direct investigations have not been carried out; for this reason, weak ternary cornplex formation cannot be eliminated as a cause for the observed poor incorporation efficiency of BIADD-labelled cysteine. It has also been observed by several investigators that non-natural amino acids with polar side chains are generally incorporated at lower yields (45, 46, 92). For exarnple, Cornish and colleagues reported that the large hydrophobic residues 7-azatryptophan, m-benzoyl-L-phenylalanine, and p-benzoyl-L- phenylalanine were inserted via non-sense suppression (vide ultra: section 3.1 -2.4) with higher efficiency than the small hydrophilic amino acids glycine. alanine. and cyclopropylglycine, or charged amino acids homoglutamate and ornithine (45, 92). There is no reason to suspect that the aminoacyl ester bond in BIADD- labelled C~S-~RNA~~*is more susceptible to chemical hydroiysis than that in ~ranscend~~tRNA or C~S-~RNA~~;therefore, deacylation (although it does occur to a significant extent during a translation reaction) is likely not the culprit. The sarne amount of labeiled tRNA was added to the translation reaction for both biotinylated tRNAs. The half-life for C~S-~RNA~in the translation reaction conditions was determined to be -30 min (discussed in chapter 1: section 1.4.4). Other factors that would contribute to no detection of BiADD-labelled luciferase in the Western blot include competition between BIADD-C~S-~RNA~~ and unlabelled C~S-~RNA~~(forrned frorn in situ aminoacylation of ~RNA'" (Le. endogenous rabbit ~RNA'~'or E cali ~RNA'~(after deacylation of BIADD-Cys- ~RNA'")) with cysteine) for the ribûsomal A site and a yield for the labelled protein that is too low to detect. A low concentration of the synthetic tRNA or low purity of the aa-tRNA stock are not likely reasons for the fack of incorporation because BAIDD-Cys- ~RNA'" and the ~ranscend~~tRNA were added to the transcription/translation reactions to the same final concentration (-1 PM) and both constructs appeared to be similar in purity (as judged by urea-PAGE in combination with a Northern blot). The possibility that the construct was incorrect (Le. the BIADD moiety was not attached specifically to the sulphur of the cysteinyl side chain but, rather, to another part of C~S-~RNA~~"(Le. the tRNA backbone or the a-amino group or both) can also be ruled out. The tR NA backbone was not labelled because no biotin, in association with unaminoa cylated ~RNA'~,was detected in the Northern blot for the labelling reaction with ~RNA'" (as oppose to C~S-~RNA'") and BlADD (chapter 2: figure 2.3A {lane c). However, as discussed in chapter 2 (section 2.4.2), the occurrence of a labelled C~S-~RNA~~~in which both the a- amino group and the cysteinyl side chah are labelled is a possibility. A doubly- labelled ~RNA'~~constmct has a very conjested 3' end and therefore rnay not be tolerated by EF-TU-GTP andfor the ribosomal A site. A biotinylated C~S-~RNA~~~ with two BIADD groups is more hydrophobie than when there is just one label; for this reason, in the HPLC chrornatogram for the fractionation of the crude BIADD- labelled C~S-~RNA~"sample (described in chapter 2: section 2.4.2), two peaks were observed which appeared to correspond to biotinylated C~S-~RNA~~~.One of the peaks (RT = 22 min) was assigned to the doubly labelled tRNA while the other peak (18 min) was identified as the elution of the correct BIADD-labelled C~S-~RNA'"in which the BIADD rnoiety is specifically attached to the cysteinyl side chain. Both tRNA constructs were used in coupled transcription/translation reactions and no biotinylated luciferase was detected. 3.4.4 Translation with IAF-labelled C~S-~RNA~~" We also tested for incorporation of the fi uorophore fi uorescein into luciferase using IAF-labelled C~S-~RNA'F(chapter 1: figure 1-16a). A coupled transcription/translation reaction was programmed with the luciferase gene and supplemented with [35~]-methionineas well as IAF-labelled C~S-~RNA~~~. Translation products were analyzed by SDS-PAGE in combination with autoradioagraphy (figure 3.1 5) as well as fluorescence imaging (not shown). Radiolabelled luciferase protein was detected at normal levels (lanes b - e); however. no fluorescent band that corresponded to Ruorescently-labelled luciferase was observed. radiolabelled + luciferase Figure 3.1 5. Autoradiographic detection of radiolabelled proteins synthesized in a 25 pL rabbît reticulocyte lysate coupled transcription/translation reaction programmed with the plasmid for luciferase and supplemented with 2 pL of [35S]-methionine(1000 Cifrnmol) and 0.5 pL (lane b); 1 pL (lane c); 1.5 pL (lane d); or 2 pL (lane e) of IAF-labelled Cys-tRNACyS(56 PM). No luciferase protein was detected when no plasmid was included in the transcriptioinl translation reaction (lane a). From each experiment, a 12 pL aliquot was analyzed. In a recent publication, Gite and CO-workersshowed that the BODIPY-FL fluorophore (figure 3.1 ) was successfully incorporated into various proteins (CC- hernolysin, dihydrofolate reductase, luciferase, chloramphenicol acetyltransferase. and bacteriorhodopsin) using 10 pL E. coli S30 extract in vitro coupled transcription/translation reactions programmed with the required genes and the N-acylaminoacyl-tRNA BOD IPY-FL-~~~~~O~~I-~RNA~~~~(figure 3.1 ) (23). The fiuorescently-labelled proteins were detected in SDS-polyacrylamide gels using a conventional UV transilluminator (similar in sensitivity to the UV mini transilluminator and phosphorimager used in our experiments) as well as a laser- based fluorescent gel scanner. BODIPY-tagged protein was observed even in transcriptionltranslation reactions containing BODIPY-FL-M~~-~RNA~at a concentration as low as 0.3 pmollpL (the concentration of IAF-labelled Cys- ~RNA'~'in one of our protein synthesis reactions was 2 pmollpL). The researchers were able to detect as Iittle as 0.35 - 0.5 ng of protein (the yield of radiolabelled luciferase in a 25 pL rabbit reticulocyte lysate coupled transcription/translation reaction supplemented with [35~]-methionineand IAF- labelled C~S-~RNA~~~was calculated to be -8 ng) . The use of N-acylated methionyl-~RNA~~'restricted the incorporation of the BODIPY-FL fiuorophore to the N-terminus (in place of the standard initiator methionine residue) of a protein whereas, luciferase could theoretically accommodate 4 fluorescein molecules at the cysteine positions. The spectral characteristics of the BODIPY-FL dye (hex = 502 nrn; Lm= 510 nm; E = 75 000) are similar to those for fluorescein (A, = 492 nm; hem = 515 nm; E = 75 000). Gite et al. determined that for every 100 protein rnolecules synthesized, there were approximately 1 - 2 protein molecules that were labelled with BODIPY-FL. The molecular fraction of fluorophore incorporated was Iimited by cornpetition for protein initiation with endogenous N- f~nn~lmethion~l-t~~~~~~~(incorporation of fluorescein into luciferase was Iimited by cornpetition for incorporation of unmodified cysteine with endogenous Cys- ~RNA~").The invesüigators showed that in general, fluorescence detection of in vitro synthesized BODIPY-FL-iabelled proteins in an SDS-polyacrylarnide gel by fluorescence irnagingr compared favourably with the sensitivity attainable using radioisotope labellingi. Gite and colleague's experiment suggests that unmodified cysteine incorporates; more efficiently than the fluorescein-labelled counterpart. Poor binding oif the fluorophore to EF-Tu-GTP's binding site can be ruled out as a possible culprit for the lack of incorporation because we have shown that IAF-C~S-~RNA~~-EF-'TU-GTPcomplexes are formed (chapter 1). Interestingly, Cornish and colleagues observed that E-dansyllysine (figure 3.3) couid not be incorporated into T4 lysozyme using an E. coli S30 extract protein synthesis system (92). The dansyl group, however, is tolerated in the binding site of EF- Tu-GTP (chapter 1). There is no reason to suspect that the aminoacyl ester bond in IAF-Cys- ~RNA'" is more susceptible to deacylation than C~S-~RNA~~~;therefore, chemical hydrolysis can be elirninated as well. The poor incorporation efficiency of IAF-labelled cysteine could be accounted for with pobor binding between the synthetic tRNA and the ribosomal A site. A list of fluorescent non-natural arnino acids which have been biosynthetically incorporated into proteins and therefore are adaptable to the ribosome is shown in figures 3.16 and 3.1 7 (34, 35, 48, 56-58, 63, 92, 93). The E-dansyllysineresidue was unsuccessfully incorporated by Cornish et ai. (92) but was successfully incorporated by Steward and colleagues (48). Both groups used the non-sense suppression rnethodology (section 3-1-2.4)- mile the latter group used an E- col; S30 extract translation system in which the release factors (which compele with the aminoacyl amber suppressor tRNA for the ribosomal A site) were denatured, the former investigators employed the same type of protein synthesis system except the release factors were not inactivated. This suggests that the E-dansyllysineside chain exhibits difficulties binding to the ribosomal A site. Fong and Vince investigated the bulk tolerance of the ribosome at the aminoacyl binding site using a series of puromycin analogues with aminoacyl side-chains which varied in structural complexity (structures shown in figure 3.6B (a - j)) and found that the two bulkiest compounds (figures 3.6B (h and i) were unable to act as acceptors of the transpeptidation reaction. This suggested that the increased bulk of the R group prevented the binding of the analogues to the ribosome (83). By analogy, the IAF-labelled cysteinyl side chain is much bulkier that the groups shown in figures 3.16 and 3.1 7 and therefore may be more difficult to accommodate sterically by the ribosomal A site. Figure 3.16. Reported fluorescent unnatural amino acids (L-configuration) that have been biosynthetically incorporated into proteins. 3.5 Summary We proposed a novel methodology for biosynthetic incorporation of non- natural amino acids into proteins (scheme 3.9). ~~stein~l-t~~~~~is exogenously prepared from E. coli RNA'", E. coli CysRS, ATP, and L-cysteine. It is then allowed to react with a thiol-reactive compound to produce a chernically- modified C~S-~RNA~~.In principle, the unnatural amino acid can then be introduced into a protein of interest via a rabbit reticulocyte lysate in vitro coupled transcription/translation reaction programmed with the gene of interest and supplemented with the synthetic tRNA. The modified cysteine would be incorporated at positions that are normally occupied by unmodified cysteine. Attempts were made to introduce fluorescein and a biotin derivative into luciferase uçing IAF-labelled C~S-~RNA~~and BIADD-labelled C~S-~RNA~~~, respectively (chapter 1: figures 1.1 6a and d, respectively). We developed conditions for the preparation of chemically-modified Cys- ~RNA'" constructs. Furthermore, we showed that reverse phase high performance liquid chromatography can be used to purify the labelled tRNAs. From the results of rabbit reticulocyte Iysate coupled transcription/translation reactions programmed with the plasmid for luciferase and supplemented with BIADD- or IAF-labelled cys-~RNA'~,it was discovered that the biotin derivative and fluorescein fluorophore were not incorporated efficient1y. 3.6 Future Directions 3.6.1 Synthesis of chemically-modified C~S-~RNA~~~with hydrophobic labels The synthetic route outlined in chapter 1 (scheme 1.4A) for alkylation of the cysteinyl side-chain of C~S-~RNA'~is restricted to the use of labelling reagents which are soluble in 40% DMSO solutions. The employment of reaction conditions in which the level of DMSO is > 40% is prevented by the presence of a 4-thiouridine base in E. coli ~RNA'~(chapter 1: figures 1.7A and 1-8C) which can react with the labelling reagent when the DMSO concentration is high. Nucleophiles such as the thiolate tend to be more reactive in polar aprotic solvents (e-g. DMSO) than in protic solvents (e-g. water) (94). This is because the anion is weakly solvated in the aprotic solvent whereas in the polar, hydrogen-bonding solvent, anions are subjected to strong interactions with the solvent molecules (94). The side-reaction with ~RNA'~'prevents the use of non- polar fluorescent labelling reagents, many of which have desirable spectral characteristics (such as long wavelengths of excitation and emission, high molar absorptivity, and high quantum yield (QY)) and are commercially available in the thiol-selective form (4). Exclusion of the use of non-polar reagents is a set-back for another major reason. Hohsaka and colleagues have investigated the adaptability of a variety of non-natural amino acids to the active center of the E. coii and rabbit reticulocyte ribosomal A sites (see section 3.1 -4.2) (57, 85). They have shown that the ribosomes accept a large variety of aromatic arnino acids (simple and complex), al! of which are non-polar. This is in line with the observation by other investigators that generally, non-polar residues are easier to incorporate than polar amino acids (45, 46, 92). For chemically-modified C~S-~RNA~~~constructs to be used as a general tool for site-specific protein labefling, it is necessary to develop reaction conditions that permit the cysteinyl side-chain to be alkylated with non-polar IabeIling reagents. A reaction route that rnay allow the synthesis of chemically-rnodified Cys- ~RNA'" constructs with non-polar reporter groups and at the same time avoids reaction with the rnodified base is shown in scherne 3.1 1 (using the thiol-reactive fluorescent labelling reagent CPM as an example). In the first step, the 4- thiouridine base of unaminoacylated E. coli ~RNA'" is reacted with iodoacetamide. The proposed conditions for this step are adapted from the Johnson et al. paper (95) and involves 10 mM Tris, pH 8.4 (HCI), 75% DMSO, 6 h incubation time, darkness, and room temperature. The protected tRNA is then precipitated with ethanol so as to remove the unreacted iodoacetamide and the other components of the reaction. In the next step, ~~stein~l-t~~~~~~is synthesized from the protected tRNA using the same conditions employed in our previous experiments (scheme 3.1 1). Johnson and colleagues have shown that ~RNA'~~with a 4-thiouridine base alkylated with 5-iodoacetamidoRuorescein still interacts with phenylaIanyl-tRNA synthetase, EF-Tu-GTP, and the ribosornal complex. The interactions are only slightly diminished compared to the s Step 1: O N O 10 mM Trls, pH 8.4 (HCI) O "c. -O-! c- -.-A 75% DMSO etc ... -0-!-O-- 0- -> O- Qj Ho O# "0 O# F) I 1--CH2CNH2 1 etc... 6 h, rt, dark protected IR NA^^' E. colt CysRS (O 04 equiv ), DU(50 equiv.), Step 2: I ATP (20 equiv ), O 50 rnM Tris, pli 7.5 (HCI), 30 rnM KCI, .------HS b 37 OC, 5 min (40 oqiiiv ) n,id~+t/sH prolecled IR NA^^' L-cysteine H ~~slein~l.1~~~~~' (prolecled) 1, IOrnM NaOCûCH,. pH 4 5 (CtifOOHi. 5 mM TCEP. 4 'C, 15 h -- -- w 2)(cH$bhN qd CPM CHb c~~.IRNA~~~ (1000 equiv.) 50 rnM Tris. pli 8 3 (HCI), 75% DMSO, O OC, 10 min Scheme 3.11. Potential route for synthesis of chemically-modified cysteinyl-tRNACYS constructs with non-polar reporter groups (using the CPM labelling reagent as an example). unmodified tRNA. By analogy, the protected RNA'" may still be an active substrate for cysteinyl-tRNA synthetase, elongation factor Tu, and the ribosome (recall that s4u is not an identity determinant for E. coli ~RNA'"). The chemically-modified C~S-~RNA'~construct with the non-polar reporter group is finally prepared by reacting the cysteinyl side chain from the protected aminoacylated tRNA with the corresponding thiol-reactive labelling reagent (scheme 3.1 1). The alkylating conditions employed are sirnilar to those used in the previous experirnents. The only difference is that DMSO is present at a 75% (as oppose to 40%) concentration. The 4-thiouridine base is blocked from reaction with the labelling reagent. The protecting group is presumed not to react with the labelling reagent because the amide group is a much poorer nucleophile than the 4-thiouridine and the thiol group of cysteine. Because the unreacted label is water-insoluble, a phenol extraction is employed to remove excess dye molecules after the labelling reaction. The labelled tRNA is recovered by ethanol precipitation. RP-HPLC with a C8 column should purify the labelled tRNA. When there is evidence that the reaction sequences in scherne 3.1 1 are successful, we intend to synthesize other chemically-rnodified C~S-~RNA~~~ constructs and test for incorporation of non-polar residues into luciferase via t~~~'?mediateNNAAM. Some labelling reagents to test are shown in figure 3.18. BODIPY FL CI-IA, BODIPY FI IA, DCIA, and iodoacetyf-LC-biotin are commercially available (Molecular Probes for the fluorophores; Pierce for the biotin derivative) while the other two (iodoadetylazobenzene and Cy5 IA) have been synthesized in our labs (Janet Kumita and Jack Zhang, respectively). The 285 BODIPY derivatives have very strong absorptivity (E = 81 000 ~-'crn-'for BODIPY FL IA and 76 000 ~-'crn"for BODIPY FL Ci-IA) and high fluorescence quantum yields (often approaching 1 in various solvents) (4) and Gite and co- workers have shown that BODIPY FL-labelled proteins synthesized biosynthetically from an in vitro coupled transcrïiption/translation system can be detected by fluorescence imaging of an SDS-PAGE gel on a UV transilluminator (23). The strong fluorescence intensity makes the fluorophores ideal candidates for studying the cellular localization and diffusion of the labelled proteins. Furthermore, fluorescence quenching, fluorescence polarization, and FRET cm be ernployed to obtain structural information as well as detect protein-protein, receptor-ligand, enzyme-substrate, and protein-membrane interactions. For FRET experiments, it has been reported that BODIPY FL is a good acceptor for CPM (see figure 3.18) (4). The dyes are also reported to be insensitive to sofvent polarity and pH and exhibit good photosfability and chemical stability between pH 3 and 10 (4). The use of BODIPY FL-labelled C~S-~RNA~~~ constructs provides an alternative method for sensitive detection of in vitro synthesized proteins, like that shown by Gite an d colleagues using BODIPY FL- labelled ~et-t~~A~~~(23).The former should be more sensitive than the latter because BODIPY-labelled cysteine can get incclrporated at multiple positions where as BODIPY-labelled methionine can onfy be introduced at the N-terminus. For the coumarin fluorophore of DClA (figure 3-18)!the emission spectrum is known to increase or decrease in intensity and shift in response to changes in the local environment (either from conformational changes in the structure of the labelled protein or changes in solvent) (4). The environment-induced spectral shifts renders the dye particularly useful for investigating protein structure, dynamics, and assembly. The coumarin dye is also known to be a good fluorescence energy acceptor from tryptophan and donor to fluorescein and the BODlPY FL molecules (4). Coumarin does not fluoresce as strongly as BODlPY FL (the molar extinction coefficient for DCIA is 31 000 ~"cm-')(4); as a result, detection of coumarin-labelled proteins synthesized from an in vitro protein synthesis system using fluorescence spectroscopy may require scaling up the transcription/translati.on reaction (vide infra: 3.7.3). Certain unnatural amino acids with a coumarin side chain have been shown to be tolerated by the E. coli protein biosynthetic machinery (18, 58). The Cy5 IA molecule (figure 3.1 8) is an important labelling reagent to test because Cy5 is an ideal molecule for single rnolecule fluorescence microscopy studies (96-1 01). Cy5 IA is actually water- soluble and therefore the original labelling conditions can be employed. The fluor is generally very fluorescent (E = 250 000 ~%m"and QY n 0.7)(1 02); therefore, detection of Cy5-labelled protein by fluorescence imaging should be possible. The biosynthetic incorporation of non-natural residues with a Cy5 side-chain has not been reported. Generation of azobenzene-labelled proteins via acetylazobenzene-labelled C~S-~RNA~~~(figure 3.18) is valuable because azobenzene can be used to photoregulate protein structure and function (103, 104). Detection of the azobenzene moieties in in vitro synthesized proteins is not as sensitive as detecting fluorescent labels and therefore will require a scale-up in the coupled transcriptionltranslations. Azobenzene can be detected by UV-Vis spectroscopy. An unnatural amino acid with an azobenzene side chain has been found to be tolerated by the E. coli translational machinery (57). Finally, the iodoacetyl-LC-biotin compound (figure 3.1 8) is a water-insoluble alternative to BIADD (see chapter 1: figure 1.16d) and is similar in structure to ~ranscend~~ tRNA (see chapter 2: figure 2-38]. If iodoacetyl-LC-biotin-modified cysteine is tolerated by the protein biosynthetic machinery, iodoacetyl-LC-biotin-labelled cys-t~~ACFwill provide a biotin-based method for detection of in vitro synthesized proteins (like the use of ~ranscend" tRNA, as marketted by Promega) and obviate the use of radiolabelled amino acids. 3.6.2 Factors that could improve incorporation efficiency One rnethod of improving the incorporation efficiency, in general, is to slow down the rate of chernical hydroiysis of the synthetic tRNA constructs. This may be achieved by lowering the incubation temperature and adding ternary complexes consisting of the chemically-modified C~S-~RNA~~~,EF-TU, and GTP rather than uncornplexed labelled tRNA to the translation reaction. The GTP- bound elongation factor slows down the rate of deacylation drastically (t1~of C~S-~RNA~~~at pH 8.3 and 4 OC = -2h, trn of C~S-~RNA~~~-EF-TU-GTPat pH 8.0 and 4 OC > -1 8h) (cf, tables 1-1 and 1.3 in chapter I). EF-1cc (the rabbit analogue of EF-Tu) is present in the lysate; however, adding preformed ternary complexes reduces the time the aa-tRNAs are exposed to the high temperature and pH of the translation reaction. Incorporation efficiency could also be improved by reducing or eliminating competition from cy~tein~1-t~~~~~~formed in situ by aminoacylation of ~RNA~" (either endogenous rabbit ~RNA'~or ~RNA'~ formed after deacylation of the synthetic tRNA or delivery of the unnatural amino acid to the ribosome) with endogenous cysteine. This can be achieved by several different ways: 1) The endogenous cysteine and ~RNA'~are rernoved; 2) The E. coIi synthetic ~RNA'" and rabbit reticulocyte lysate coupled transcriptionftranslation system are switched to a different t~~~~~~/ribosomepair such that the deacylated labelled ~RNA'~is not recognized by any of the endogenous aaRS (some pairs to try include E. coli t~N~'rj/wheatgerm ribosome and rabbit ~RNA~~IE.coli ribosome); and/or 3) The use of chemically-modified C~S-~RNA~~~is replaced by the employment of chemically-modified C~S-~RNA~'*(discussed in section 3.1.5) (recall that E. coli ~RNA"* is aminoacylated 5 - 8-fold less efficiently than the natjve tRNA by E. coli CysRS). 3.6.3 Cell-free production of rnilligram quantities of labelled proteins Low yields of cell-free protein synthesis systems such as the rabbit reticulocyte lysate, E. coli S-30 extract, and wheat germ extract have hindered the application of non-natural amino acid mutagenesis. For exarnple, a 50 pL rabbit reticulocyte lysate coupled transcription/translation reaction typically yields 150 -500 ng of protein (Promega technical bulletin TB126). A 50 pL reaction with the E. coli S-30 extract is able to synthesize 50 - 250 ng of protein. From a reaction with the wheat germ extract, the yield is approximately 150 - 500 ngf50 PL. Biochemical and biophysical techniques (e.g. X-ray crystallography and NMR) generally require much larger quantities of proteins. 289 Kigawa and colleagues have recently reported a strategy for improving the productivity of the E. coli 5230 extract protein synthesis system (105). Three major modifications were made. The first modification involved a replacement of the energy source regenerating system. Typially a phosphoenol pyruvate/pyruvate kinase (PEPIPK) system is emplo~ed.However, a PEPIPK system has an inhibitory effect on protein synthesis. The investigators tested the efficiency of protein synthesis when an acetyl plhosphatelacetyl kinase (APIAK) or a creatine phosphatelcreatine kinase t(CP1CK) (usually used in eukaryotic cell-free systems) system was used instead o~ the PEPiPK pair. It was discovered that protein productivity in the three different systems decreased in the order corresponding to CPICK > APIAK > PEPIPK From the results, the traditionally used PEPIPK system was substituted for -the CPICK system in the E. coli 530 extract coupled transcription/transIation sys~tern. The concentrations of polyethylene glycol (PEG), magnesium ions, and other components of the traenscriptionltranslation reaction were optimized as well. To increase productivity, Kigawa and CO-workersalso condensed the S-30 extract. The condensation vas achieved by dialysis against a PEG-containing solution, a procedure whicht was simple and amenable to scale up. It was discovered that S-30 extracd which was condensed 2 - 2.5-fold appreciably increased both the initial rate of protein synthesis and the total amount of synthesized protein. The third and last modification that Kigawa et al. made to improve productivity was to cany out the transcription/translation reaction in conjunction with a disposable dialyzer. The protein synthesis system was dialyzed against a 1O-fold volume of a buffer containing al1 components of the reaction except the creatine kinase, the plasmid vector, the T7 RNA polyrnerase, the S30 extract, and the ribonuclease inhibitor. When all three aforementioned modifications (in addition to supplements of plasrnid vector and T7 RNA polymerase as well as an exchange of the external dialysis solution) were incorporated into the E. coli S30 extract coupled transcription/translation reaction, it was observed that as much as 6 mg of protein was synthesized per mlof reaction mixture in 21 h, a yield that is 1000 times higher than that obtained from the standard situation. The high productivity of the improved cell-free protein synthesis system is comparable to the yield from the in vivo expression methods. The high protein productivity from the method reported by Kigawa and co- workers provides a major reason for us to switch to using the E. coli S-30 extract instead of the rabbit reticulocyte lysate for in vifro coupled transcriptionltranslation reactions. In doing so, the synthetic tRNA species must be switched as well to one that is not of E. coliorigin. Alternatively, the use of labelled C~S-~RNA'"is substituted by the use of labelled C~S-~RNA"~~. 3.6.4 t~~~~~~-rnediatedmutagenesis for introduction of multiple fiuorophores and for studies on membrane protein structure Incorporation of more than one type of fluorescent probe is necessary for FRET experirnents and simultaneous structural studies of different parts of a protein. The use of chemically-modified C~S-~RNA~~~does not eoable introduction of more than one type of fiuorophore into the target protein in a controlled manner; that is, random labelling of the target protein occurs when two different labelled tRNA constructs are added to a coupled transcriptionftranslation reaction, as dictated by the adaptability of each of the unnatural amino acid to the protein biosynthetic machinery. This limitation, however, could be circumvented by using two or more different NNAAM techniques at the same time. 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FEBS Le# 442, 15-1 9 Appendix A: Buffer Compositions Chapter 1 buffer A IO MM potassium phosphate, pH 6.8 (KOH), 1 mM PME, 10% glycerol buffer B 50 mM NaH2P04, pH 8.0 (NaOH), 300 mM NaCI, 10 mM imidazole buffer C 50 mM Tris, pH 7.5 (HCI), 50 mM NH4C1, 50 mM KCI, 10 mM MgCl*, IO mM PME, 50 pM GDP buffer D 50 mM NaH2P04, pH 8.0 (NaOH), 300 mM NaCI, 500 mM imidazole buffer E 50 mM Tris, pH 7.5 (HCI), 30 mM KCI, 10 mM MgCl2 buffer F 50 mM Tris, pH 7.6 (HCI), 60 mM NH4CI, 7 mM MgCI2, 7 mM PME, 15% glycerol buffer G 50 mM Tris, pH 8.0 (HCI), 60 mM NH4CI, 7 mM MgC12, 7 mM PME, 15% glycerol, 5 PM GDP buffer H 50 mM Tris, pH 7.0 (HCI), 60 mM NH4C1, 7 mM MgC12, 7 mM PME, 15% glycerol, 5 pM GDP buffer l 50 mM Tris, pH 7.0 (HCI), 60 mM NH4C1, 7 rnM MgCl;>, 7 mM PME, 15% glycerol, 80 mM imidazole, 40 pM GDP buffer J IO NaOCOCH3, pH 4.5 (CH3COOH), 5 mM TCEP buffer K 50 mM Tris, pH 7.5 (HCI), 50 mM NH4CI, 50 mM KCI, 10 mM MgC12, 1 rnM GTP, and 5 mM PME buffer L 50 mM Hepes, pH 7.5 (NaOH),150 mM NaCI, 50 mM NH&I, 10 mM MgC12, 5 mM PME, and 50 pM GTP Appendix A: Buffer Compositions Chapter 1 buffer A 1O mM potassium phosphate, pH 6.8 (KOH), 1 mM PME, 10% glycerol buffer B 50 mM NaH2P04, pH 8.0 (NaOH), 300 mM NaCl, IOmM imidazole 50 mM Tris, pH 7.5 (HCI), 50 mM NH4CI, 50 mM UCI, 10 mM MgC12,IO mM PME, 50 pM GDP buffer D 50 mM NaH2P04, pH 8.0 (NaOH), 300 mM NaCI, 500 mM imidazole buffer E 50 mM Tris, pH 7.5 (HCI). 30 mM KCI, 10 mM MgCl2 buffer F 50 mM Tris, pH 7.6 (HCI), 60 mM NH4CI, 7 mM Mg&, 7 mM PME, 15% glycerol buffer G 50 mM Tris, pH 8.0 (HCI), 60 mM NH4CI, 7 mM MgC12, 7 mM PME, 15% glycerol, 5 pM GDP buffer H 50 mM Tris, pH 7.0 (HCI), 60 mM NH4CI, 7 mM MgCI2, 7 mM PME, 15% glycerol, 5 MM GDP buffer 1 50 mM Tris, pH 7.0 (HCI), 60 mM NH4CI, 7 mM MgCI2, 7 mM PME, 15% glycerol, 80 mM imidazole, 40 FM GDP buffer J 10 NaOCOCH3, pH 4.5 (CH3COOH), 5 mM TCEP buffer K 50 mM Tris, pH 7.5 (HCI), 50 mM NH4CI, 50 mM KCI, IOmM MgCI2, 1 mM GTP, and 5 mM PME buffer L 50 mM Hepes, pH 7.5 (NaOH),ISO mM NaCI, 50 mM NH4CI, 10 mM MgCI2, 5 mM BME, and 50 pM GTP