Wobble Modifications and Other Features in Transfer RNA Important for Decoding and Reading Frame Maintenance

Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance

Joakim Näsvall

Department of Molecular Biology Umeå University Umeå, 2007 Department of Molecular Biology Umeå University SE-901 87 Umeå, Sweden

Front cover: Cartoon representation of a tRNA molecule see legend of Figure 2B (p. 5) for details.

Copyright © 2007 by Joakim Näsvall ISBN 978-91-7264-437-3 Printed by Print & Media, Umeå University, Umeå, 2007 TABLE OF CONTENTS MAIN REFERENCES 1 ABSTRACT 2 1 INTRODUCTION 3 1.1 The genetic code 3 1.2 Transfer RNA 4 1.2.1 Modified nucleosides in transfer RNA 5 1.3 Translation 8 1.3.1 Translational errors 11 1.3.2 Proofreading during protein synthesis 12 1.4 Decoding 13 1.4.1 The Wobble Hypothesis 13 1.4.2 The Revised Wobble Rules 15 1.4.3 The “Two out of three” Hypothesis 17 1.4.4 The decoding center: The ribosomal A-site 18 1.5 Reading frame maintenance 21 1.5.1 Frameshift suppression 21 1.5.2 Quadruplet translocation and out of frame binding 22 1.5.3 P-site slippage and the “Dual Error” model 23 1.5.4 The ribosomal P-site 26 2 RESULTS AND DISCUSSION 28 5 2.1 Synthesis of cmoP U34P in tRNA (Paper I) 28 5 2.2 Function of cmoP U34P in decoding (Paper I and II) 31 2.3 A dominant frameshift suppressor accumulates novel modified nucleosides in tRNA (Paper III) 37 2.4 The “ribosomal grip” of the peptidyl-tRNA is critical for reading frame maintenance? (Paper IV) 40 3 CONCLUSIONS 43 ACKNOWLEDGEMENTS 44 REFERENCES 45 PAPERS I-IV

MAIN REFERENCES

This thesis is based on the following papers and manuscripts, which are referred to in the text with their roman numerals (Paper I – IV):

I. S.U Joakim Näsvall,U Peng Chen and Glenn R. Björk, (2004). The

Pro modified wobble nucleoside uridine-5-oxyacetic acid in tRNA cmo5UGG promotes reading of all four proline codons in vivo. RNA 10: 1662- 1673.

II. S.U Joakim Näsvall,U Peng Chen and Glenn R. Björk, (2007). The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons. RNA Published online Oct. 17, 2007

III. Peng Chen, Pamela F. Crain, S.U Joakim Näsvall,U Steven C. Pomerantz and Glenn R. Björk, (2005). A “gain of function” mutation in a protein

mediates the production of novel nucleosides. The EMBO Journal 24T :T 1842 – 1851

IV. S.U Joakim Näsvall U and Glenn R. Björk, (2007). The “ribosomal grip” of the peptidyl-tRNA is critical for reading frame maintenance? MANUSCRIPT

The papers are reprinted with permission from the copyright owners.

1 ABSTRACT

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA 5 recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmoP U34),P found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. 5 To study the function of cmoP U34P we have identified two genes, cmoA and cmoB, which are 5 required for the synthesis of cmoP U34P in tRNA. We have shown that the proline, alanine and 5 valine tRNAs containing cmoP U34P are capable of reading codons ending with any of the four 5 nucleotides, while the threonine tRNA is not, and the importance of having cmoP UP is 5 different for the different tRNAs. In addition, we found that cmoP UP is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln),

Pro when tRNAcmo5UGG occupies the P-site. sufY mutants accumulate novel modified 5 2 nucleosides at the wobble position of tRNAs that should normally have (c)mnmP sP P U34.P The

Gln presence of an extra sidechain (C10B HB 17B )B on the wobble nucleoside of tRNA cmnm5s2UUG leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site

Pro peptidyl- tRNA cmo5UGG to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding

Pro tRNA cmo5UGG . The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

2 1 INTRODUCTION

1.1 The Genetic Code

Shortly after the discovery of the structure of DNA, Crick et al provided genetic evidence suggesting that each amino acid encoded in the genetic message is represented by a group of three (or, less likely, by a multiple of three) nucleosides (Crick et al. 1961). They also hypothesized that the code words (codons) were not overlapping and that there was no punctuation separating consecutive codons. With a triplet nature of the code the number of possible codons is 64, which led Crick et al to predict that the code is degenerate in the sense that each of the twenty amino acids may be represented by more than one codon (Crick et al. 1961). By the work of mainly the groups led by Marshall Nirenberg and Gobind Khorana, the code was deciphered a few years later (reviewed in (Nirenberg 2004)) (Figure 1). Codons starting with the same nucleosides in the first two positions are referred to as a codon box. The eight boxes where all four codons encode the same amino acid are referred to as family (or four-fold degenerate) codon boxes (shaded in Figure 1) and the remaining eight boxes (white in Figure 1) are referred to as mixed (two- or three-fold degenerate) codon boxes.

3 Second letter [N(II)] UCAG Ph e (F) Tyr ( Y) Cys (C) U C U Ser (S) St o p A Leu (L) St o p Tr p ( W ) G Third letter [N(III)] U His (H) C C Leu (L) Pr o (P) Ar g (R) A Gln (Q)

er [N(I)] G t t Asn (N) Ser (S) U A Ile (I) Thr (T) C A irst le irst Lys (K) Arg (R) F Met (M) G Asp (D) U G Val ( V) Al a (A) Gly (G) C Glu (E) A G Figure 1. The genetic code. Only the twenty standard amino acids are included. The eight family codon boxes are shaded.

1.2 Transfer RNA

Before the nature of the code was known, Francis Crick hypothesized that an adaptor molecule was needed in order to translate the information in mRNA into the correct amino acid sequence (Crick 1955). This adaptor was later found by Hoagland et al to be a small soluable RNA (Hoagland et al. 1958), later named transfer RNA (tRNA). tRNAs are between 76 and 95 nucleotides in length and are often represented by the “clover-leaf” model, showing the secondary structure formed by base pairing between different parts of the sequence (Figure 2A). In the three dimensional structure, first solved by the groups led by Alexander Rich (Suddath et al. 1974) and Aaron Klug

4 (Robertus et al. 1974), tRNA forms an L-shaped structure with the anticodon in the end of one arm and the acceptor stem, where the aminoacid is attached, in the other arm (Figure 2B).

Figure 2. A: Secondary structure of tRNA (“clover-leaf” model). Broken lines indicate regions of variable length. The numbering start in the 5’-end and skips variable regions. B: Cartoon representation of the crystal structure of a tRNA. The image was made with PyMOL (DeLano 2002) from the crystal structure of yeast phenylalanine tRNA, pdb-code: 1EHZ (Shi & Moore 2000).

1.2.1 Modified nucleosides in transfer RNA

In addition to the canonical nucleosides, tRNA contains a number of modified nucleosides. These are derivatives of the four normal nucleosides and are synthesized on the tRNA by specific enzymes. Some of the modifications are close to universally conserved throughout the three domains of life (Bacteria, Archaea and Eukarya), while others are specific to the different domains or

5 subgroups within the individual domains (Björk & Hagervall 2005). The anticodon loop is frequently modified and contains a wide variety of different modifications, especially at position 34 (the wobble position) and position 37 (immediately 3’ of the anticodon) (Figure 3 and Figure 4).

76 1

Ψ D 4 m1 A Ψ sU 8 17 Gm 55

20 45 Ψ D 5 acp 3 U m U m1 A D m7 G Ψ s2 C, Um, Cm, Ψ m 2 A, m 66 A, i A, 34 36 35 ms22 i 66 A, ms io A, Anticodon t 6 A, m66 t A, m 1 G cmo55 U, mcmo U, mnm5 s 2 U, mnm55 U, cmnm s2 U, cmnm5 Um, cmnm 55 s2 U, mnm se2 U, k 2 C, Cm 4 ac C, Q, GluQ, I, Gm Figure 3. Locations of modifications found in tRNA of Salmonella enterica (Björk & Hagervall 2005).

6 O O O O NH2 H C O 3 NH N CH3 HO NH NH N N

N O N S N N O N NH2 HO HO HO HO O O O O

OH OH OH OH OH OH OH O CH3

Uridine-5-oxyacetic acid 5-methylaminomethyl-2-thiouridine 1-methylguanosine 2'-O-methylcytidine cmo 5 U mnm 52 s U m1 G Cm Figure 4. Stuctures of four modified nucleosides found in the anticodon loop of tRNA. The square boxes highlight the differences compared to the canonical nucleosides.

The modified nucleosides outside the anticodon stem and loop are expected to influence tRNA function mainly through their effect on structure and stability 5 of tRNA. mP U54P (5-methyluridine, ribothymidine) and Ψ55 (pseudouridine) are present in almost all tRNAs in most organisms. These are involved in 5 tertiary interactions with other nucleosides (mP U54P to A58 and Ψ55 to G18) and have been shown to contribute to the thermal stability of tRNA (Davanloo et al. 1979; Kinghorn et al. 2002). Mutants lacking these modifications are viable and have surprisingly mild phenotypes (Björk & 4 Neidhardt 1971, 1975; Urbonavicius et al. 2002). sP U8P absorbs light in the near-UV spectrum, causing it to form a photo-crosslink to C13. Presence of this cross-link in tRNA leads to growth arrest, which partially protects the cell from accumulating UV-induced mutations (reviewed in (Björk & Hagervall 2005)). The modifications present in the anticodon stem and loop influence the structure and flexibility of the anticodon loop, thereby influencing the decoding process. The nucleosides at position 32 and 38 of tRNA are involved in a bifurcated hydrogen bond (Auffinger & Westhof 1999). The bases at these positions appear to have co-evolved with their anticodons to ensure uniform binding of all tRNAs to their respective codons, by

7 influencing the affinity of the anticodon stem-loop to the ribosomal A-site. Neither the mechanism behind this effect nor how modifications affect it is known (Olejniczak & Uhlenbeck 2006). Mutations affecting modifications at position 32, 37 and 38 have been shown to lead to decreased rate of translation at codons read by affected tRNAs, and to influence reading frame maintenance (Björk & Hagervall 2005; Björk et al. 1989; Urbonavicius et al. 2001). Some modifications at position 37 (3’ next to the anticodon) and 34 (the first position of the anticodon) have been shown to pre-structure the anticodon loops of ASLs in solution and to increase or enable the binding of ASLs to codons in the ribosomal A-site (Murphy et al. 2004; Stuart et al. 2003; Yarian et al. 2002). In one case the identity of a tRNA is completely changed by introduction of a single modification: An isoleucine tRNA with 2 the modified cytidine derivative kP CP (lysidine) at position 34 reads only AUA (Ile) codons and is aminoacylated with isoleucine, but without the modification it reads AUG (Met) and is aminoacylated with methionine (Soma et al. 2003).

1.3 Translation

The bacterial ribosome is a large ribonucleoprotein complex composed of approximately two thirds RNA and one third protein. The small subunit (30S) is made up of 16S rRNA and 21 different proteins, while the large subunit (50S) is made up of 5S and 23S rRNA and 31 or more proteins (Noller & Nomura 1996).

8 Translation in bacteria is initiated by formation of an initiation complex by Met the binding of fMet-tRNAP P to the start codon of the mRNA in the peptidyl site (P-site) of the 30S subunit, a process requiring the three translation initiation factors IF1, 2 and 3 (reviewed by (Gualerzi et al. 2000)). The initiation complex associates with a 50S subunit, leading to the formation of a 70S ribosome with the second codon of the mRNA in the aminoacyl site (A- site). Translation elongation can be divided into several different steps (Figure 5). First a ternary complex of aminoacyl-tRNA (aa-tRNA), elongation factor Tu (EF-Tu) and GTP binds to the ribosome, placing the anticodon of the tRNA in the A-site, while the acceptor stem carrying the amino acid is bound to EF-Tu. If the proper codon-anticodon interaction is formed during initial selection, the GTP of the ternary complex is hydrolyzed, leading to release of EF-Tu from the ribosome. After this the aa-tRNA is accommodated into the A-site, which results in the positioning of the acceptor stem with its amino acid in the peptidyl transfer center of the 50S subunit (reviewed in (Rodnina et al. 2005)). The peptide is transferred from the P-site peptidyl- tRNA to the amino acid on the A-site aa-tRNA, leading to extension of the peptide by one amino acid residue.

9 GDP GTP aa-tRNA EF- Ts

EF- Tu * GTP Ter n ar y Co m p l ex EF- Tu * GDP

In EPA it Se ial g lec in ti d on ea fr o ro P

Deacylated tRNA EPA EPA

EF- G* GDP P n e o T p i r t at GTP a id c n y o s l sl fe n r a Tr

EPA GDP EF- G* GTP Figure 5. The translation elongation cycle. Adapted from (Kurland et al. 1996).

Elongation factor G (EF-G) facilitates the translocation, resulting in the coordinated movement of first the acceptor stems and later the anticodon stem-loops of the P-site deacylated tRNA into the exit site (E-site) and the peptidyl-tRNA (still paired to its codon in mRNA) from the A-site to the P- site, leading to a new codon being exposed in the now empty A-site (Zavialov & Ehrenberg 2003). Translation is terminated when a release factor (RF1 or RF2) recognizes a stop codon in the A-site, leading to hydrolysis of peptidyl-

10 tRNA and dissociation of the ribosomal subunits from each other and from the mRNA (see (Kisselev et al. 2003) for review).

1.3.1 Translational Errors

When the ribosome translates a message it is important that the correct amino acid is incorporated at each codon and that translation is carried out in the correct reading frame. There are two general types of translational errors that may occur (Reviewed in (Engelberg-Kulka & Schoulaker-Schwarz 1996; Kurland et al. 1996)): Processivity errors are errors that lead to production of truncated or too long

peptides. This may happen in several different ways; PrematureU termination U

and drop-offU U is when the ribosome dissociates from the mRNA with or

without the involvement of a release factor, respectively. StopU codon read-

through U is when translation continues beyond a stop codon, usually caused by

a tRNA recognizing the stop codon. FrameshiftingU U is when the ribosome fails to maintain the correct reading frame and ends up in a different frame. Frameshifting will be discussed further later in this thesis. Missense errors are errors that result in incorporation of the wrong amino acid. This can happen in two different ways: Either a mis-charged tRNA recognizes its cognate codon in the ribosomal A-site, or, more commonly, a correctly charged tRNA recognizes a near-cognate codon that specifies a different amino acid.

11 1.3.2 Proofreading During Protein Synthesis

To ensure that missense errors do not occur too often, there are several levels of proofreading before the formation of the peptide bond. The first few levels of proofreading takes place in the specific aa-tRNA synthases (aaRS:es). Each aaRS is highly specific for its cognate amino acid and tRNA. In the cases when aaRS:es have difficulties in discriminating their cognate amino acid from another amino acid, they have specific editing domains that hydrolyzes misacylated aa-tRNAs or aa-AMPs (Ibba & Soll 1999; Jakubowski & Goldman 1992). The next level of proofreading is on the level of formation of the aa-tRNA•EF-Tu•GTP ternary complex. The tRNAs have coevolved with EF-Tu in such a way that if the amino acid bind strongly to the amino acid binding pocket of EF-Tu, the acceptor stem of the tRNA will bind weakly to EF-Tu, ensuring that all correctly acylated tRNAs bind with similar affinity to EF-Tu (LaRiviere et al. 2001). The above mechanisms ensure that misacylated tRNAs are very rarely used by the ribosome. The final level of proofreading occur during decoding in the ribosomal A-site, where the anticodon of the tRNA associates with the codon in mRNA and the codon-anticodon complex is tested through interactions with the ribsome, ensuring that codons are not read by the wrong tRNAs (Rodnina et al. 2005).

12 1.4 Decoding

1.4.1 The wobble hypothesis

When the genetic code was almost completely known and the first few tRNAs were sequenced, it was realized that there does not have to be one specific tRNA recognizing each codon. With his wobble hypothesis, Francis Crick predicted that while the first two positions of the codon-anticodon interaction have to form strict Watson-Crick pairs, the ribosome would allow a few alternative base pairs in the third position (Crick 1966). According to this model, an Inosine (I, a deaminated A) in the wobble position (position 34) of tRNA could form basepairs with U(III), C(III) and A(III) in the codon, G34 could pair with C(III) and U(III), and U34 could pair with A(III) and G(III) (The roman numeral III within parenthesis indicate the third position of the codon)(Figure 6 and Table 1). Compared to a Watson-Crick pair, either the uridine in the predicted G34-U(III) and U34-G(III) pairs is displaced towards the major groove of the codon-anticodon complex or the guanine is displaced towards the minor groove.

13 O H N O H N N O H N U N N N H N N N H O R R G CGR N N N N H O R N H H H

O H N NH O N O H N U N N N H N N N H O R R A UIR N N N O R

H H N O H N N O H N N

N N H N N N H N N R ICR IAR N N N N O R Figure 6. Structure of the base pairs allowed according to the wobble hypothesis (Crick 1966). The “–R”s show the location of the C1’ atoms of the ribosyl moieties. The three pairs to the left are in normal Watson-Crick geometry.

At the time Crick presented his wobble hypothesis, the only known modified nucleoside in the anticodon of tRNA was I34. Later it has been found that uridine at the wobble position of tRNA is almost always modified (summarized in (Sprinzl & Vassilenko 2005)). In eubacterial tRNA there are two classes of modified uridines found at the wobble position. One class 5 (xoP U),P which is found exclusively in family codon boxes, consists of the

14 5 related nucleosides uridine-5-oxyacetic acid (cmoP UP (Figure 5), present in 5 Salmonella enterica and Escherichia coli) and 5-methoxyuridine (moP U,P present in Bacillus subtilis). Different modifications of the other class 5 2 (xmP (sP P )U)P are found in tRNA reading codons in the mixed codon boxes and the glycine family codon box in tRNA from all three domains of life (Björk & Hagervall 2005; Huang et al. 2005; McCloskey et al. 2001).

1.4.2 The Revised Wobble Rules

Yokoyama et al measured the relative stability of two conformations (C2’- endo and C3’-endo, Figure 7) of the ribosyl moiety of nucleosides and 5 nucleotides of uridine and the different classes of modified uridines (xoP UP 5 2 and xmP (sP P )U)P found in the wobble position of bacterial tRNAs (Yokoyama et al. 1985).

H2’ H3’ C5’ B B C5’

O2’ O3’ H3’ H2’ H4’ H1’ H4’ H1’

O3’ O2’

C2’-en d o C3’-en d o Figure 7. Alternative conformations of the ribosyl moiety of a nucleoside. “B” is the base.

15 5 Their data showed that the equilibrium for the xoP UP class of modified nucleosides was shifted towards the 2’-endo form through an interaction between the modification and the 5’-phosphate. If the ribose was in the 2’- endo form it would give the wobble uridine enough reach to form a base pair with U in mRNA, but an interaction with C would be impossible through this mechanism (Table 1). The shift in equilibrium compared to U was seen for 5 5 5 both moP UP and cmoP U,P but not with hoP UP (5-hydroxy uridine), indicating that 5 hoP UP (which is normally not found in tRNA) would have a codon recognition pattern very similar to uridine (Yokoyama et al. 1985). The other class of 5 2 uridine modifications, xmP (sP P )U,P shifted the equilibrium in the other direction, towards the 3’-endo conformation. According to the model, the function of 5 2 the xmP (sP P )UP modifications would be to restrict the wobble uridine from interacting with pyrimidines in the mRNA, thereby preventing misreading of the pyrimidine-ending codons in the mixed codon boxes. As a consequence to this restricted wobble, tRNAs carrying this modification would also be poor at reading G-ending codons (Yokoyama et al. 1985) (Table 1). Table 1. Revised Wobble rules Anticodon N34 Codon N(III)

U A G C G I U C A G C U 5 2 xmP (sP P )UP A (G) 5 xoP PU A G U The rules for U, C, I and G are from Crick’s original Wobble Hypothesis (Crick 1966). The rules for modified uridines are from the revised wobble rules (Yokoyama et al. 1985).

16 In contrast to the model it has been shown that hypo-modification of the Lys 5 2 wobble nucleoside in tRNAP ,P normally containing mnmP sP P U34,P leads to decreased mis-incorporation of lysine at AAU/C asparagine codons (Hagervall et al. 1998) and also that modification of the wobble position is required for binding to AAG (Lys) codons by an anticodon-stem-loop Lys 5 2 (ASLP )P (Yarian et al. 2002). Thus, there are data showing that xmP (sP P )UP modifications improve, rather than restrict wobble towards G-ending codons and, to some extent, even towards pyrimidine-ending codons. This indicates that the prediction for this type of uridine modification may be wrong and perhaps that the relative stability of the 2’- vs. 3’-endo forms of the ribose of the wobble nucleoside is not the determining factor for its ability to wobble.

1.4.3 The “Two out of three” Hypothesis

The two above models (the wobble hypothesis and the revised wobble rules) are both based on the assumption that the wobble pair [N34-N(III)] has to be able to make at least two neighboring hydrogen bonds in order to be stable enough to be accepted by the ribosome (Crick 1966; Yokoyama et al. 1985). In an alternative model, Lagerkvist proposed that in certain cases there is no need for a stable interaction between position 34 of tRNA and the third position of the codon. According to this model “third position misreading” have a high probability to occur with two G-C pairs in the first two positions of a codon-anticodon complex and a low probability to occur if the first two positions form only A-U pairs (Lagerkvist 1978). The model was supported by an in vitro translation system where some tRNAs could read codons where they were not predicted to be able to form interactions in the wobble position. Later, the discovery that Mycoplasma and mitochondria use only tRNAs

17 containing unmodified uridine at the wobble position to decode all four codons in some or all of the family codon boxes provided more support for this model. Later it was found that by changing the nucleotide at position 32

Gly of mutant variants of E. coli tRNA CCC to resemble the anticodon loop of

Gly Mycoplasma tRNA UCC allowed them to read all four glycine codons (Borén et al. 1993; Claesson et al. 1995; Lustig et al. 1993). Recently this have been shown to be caused by an effect of the 32-38 base pair on the binding to the ribosomal A-site (Olejniczak & Uhlenbeck 2006), providing a possible explanation for the apparently less efficient discrimination at the third position by these tRNAs.

1.4.4 The decoding center: The ribosomal A-site

Several high resolution crystal structures with tRNAs or ASLs interacting with their cognate codons in the ribosomal A-site have been published (Murphy & Ramakrishnan 2004; Murphy et al. 2004; Ogle et al. 2001; Selmer et al. 2006). From these structures it is clear that during the decoding 1 process a cognateTPF F PT codon-anticodon interaction is recognized by an induced fit mechanism, involving three universally conserved residues of 16S rRNA: A1492, A1493 and G530 (Figure 8). A1493 and A1492 reach into the minor groove of the codon-anticodon minihelix and make hydrogen bonds to the first two positions of the codon-anticodon interaction. These interactions can

1 TP PT “Cognate” in this thesis means the codon-anticodon complex forms Watson-Crick base pairs in the first two positions and an interaction in the third position that is allowed according to Crick’s wobble hypothesis. “Near-cognate” means there is a mismatch at one of the first two positions, or the pair at the third position is not predicted to be allowed according to Crick, even in the cases when a third position mismatch would result in incorporation of the correct amino acid.

18 only occur if the base pairs are in Watson-Crick geometry. G530 contributes by forming hydrogen bonds to both A1492 and the wobble position of the codon. In the wobble position of the codon-anticodon complex there are some restrictions, but the interactions with the ribosome still allows the pair to deviate from Watson-Crick geometry (Ogle et al. 2001). During initial selection (Figure 5) the anticodon stem of aa-tRNA is bent by interactions with EF-Tu, the ribosome and the codon. If the codon-anticodon complex is recognized as cognate through the above induced fit mechanism, the rate of activation of the GTPase activity of EF-Tu is increased. After hydrolysis of GTP, EF-Tu•GDP dissociates from the ribosome. This releases the acceptor stem of the aa-tRNA, so that the strained structure can relax, leading to movement of the acceptor stem by close to 70 Å into the peptidyl transfer center of the 50S subunit (Accomodation). During this movement, near- cognate aa-tRNAs that passed through the initial selection step have a higher rate of dissociation and a lower rate of accommodation than cognate aa- tRNAs. In summary, non-cognate aa-tRNAs does not pass through the initial selection step, whereas two steps of discrimination are needed to reduce decoding by near-cognate aa-tRNAs to an acceptable level (Reviewed in (Rodnina et al. 2005)).

U35 U36

U34

C1054 G530 A1493 A1492

Mg2+

A(III) A(I) A(II)

Figure 8. The ribosomal decoding center containing a cognate codon-anticodon complex. Dashed lines indicate hydrogen bonds between the codon-anticodon complex and 16S rRNA residues, and coordination of a magnesium ion by G530 and the codon wobble position. The magnesium ion is coordinated by two more ribosomal residues not shown in the image. tRNA and mRNA residues are shown with dark grey carbons, rRNA with light grey carbons.The picture was rendered with Lys PyMOL (DeLano 2002) from the crystal structure of ASLUUU bound to an AAA codon in the 30S A-site (Murphy et al. 2004) (pdb code: 1XMQ).

20 1.5 Reading Frame Maintenance

The reading frame is initially set during translation initiation, where after it is faithfully maintained for hundreds or even more than a thousand codons with only occasional errors. If a translating ribosome shifts frame and continue translation in the wrong frame, the information in the message is lost, as the new frame will encode an incorrect amino acid sequence and usually contains stop codons. It has been estimated that the reading frame is lost at a frequency -5 of only about 10P P per codon. At certain sequences frameshifting can occur more often, due to features in the mRNA sequence or structure where reading frame maintenance is less efficient. In some cases such sites have been selected through evolution to become regulatory devices, which are used to regulate the rate of synthesis of a protein or to set a ratio between two different products produced from overlapping reading frames in the same gene. Examples of such programmed frameshifting can be found in the gene encoding peptide release factor 2 (RF2) in bacteria and in the expression of the gag and pol proteins of retroviruses such as HIV and the TY retrotransposons of yeast (Reviewed in (Farabaugh 1996) and (Farabaugh & Björk 1999)).

1.5.1 Frameshift suppression

Mutations that leads to loss of the reading frame of the gene they occur in are referred to as frameshift mutations. This type of mutations can be rescued by an additional mutation within the same gene that restores the original reading frame (Crick et al. 1961). Another way to rescue a frameshift mutant, at least partially, is by mutations affecting components of the translational machinery

21 so that ribosomes can be repositioned into the correct frame (Reviewed in (Atkins & Gesteland 1995; Atkins et al. 2000) and (Roth 1981)). This latter group of mutants are referred to as frameshift suppressors. Many of the characterized frameshift suppressors have mutations in tRNA genes, others in genes encoding tRNA modification enzymes or other components of the translational machinery. A +1 frameshift suppressor is a suppressor that can shift the ribosome one base forward, whereas -1 frameshift suppressors can cause the ribosome to shift one base backwards.

1.5.2 Quadruplet translocation and out of frame binding

Some of the first characterized +1 frameshift suppressors were insertions of single nucleotides in the anticodon loops of proline (sufA and sufB) and glycine tRNAs (sufD) (Riddle & Carbon 1973; Riddle & Roth 1970). One model for the mechanism of suppression by these mutant tRNAs was that they would recognize a four base codon instead of the normal three base codon in the A-site of the ribosome, inducing a four base translocation. According to this model the triplet nature of the code is set by the anticodon in tRNA acting as a “yard stick” that determines the length of translocation. The efficiency of suppressors of this type to suppress different sites appear to be correlated to the level of complementarity between the “four base anticodon” and the “four base codon”, but there is no absolute requirement for an ability to form a base pair in the fourth position (Gaber & Culbertson 1984; Magliery et al. 2001; Tuohy et al. 1992). As a variation on this theme, Curran and Yarus presented a model where the oversized anticodon loop has its effect in the P-site, by moving the boundary between the A-site and the P- site in such a way that the next tRNA can bind to a three base codon in the +1

22 frame and subsequently induce a quadruplet translocation (Curran & Yarus 1987). These models require either that the A-site has room for an extra base pair in the codon-anticodon complex, or that the position of the decoding centre of the ribosome is flexible enough to accept out of frame binding of an incoming tRNA in the A-site.

1.5.3 P-site slippage and the “Dual Error” model

There are frameshift suppressors that can not be explained by quadruplet translocation, as the tRNAs responsible for frameshifting have normal-sized anticodons (Farabaugh & Björk 1999; Qian et al. 1998; Tuohy et al. 1992). An alternative model (the “dual error” model, Figure 9) states that, while the A-site is empty, a peptidyl-tRNA occupying the P-site may slip forward relative to the mRNA and re-pair in the overlapping +1 frame. This normally happens at a very low frequency, but if the peptidyl-tRNA in the P-site is somehow abnormal (hypo-modified, mutant or near-cognate to the codon) it may have an increased propensity to shift frame (the first error in this model is when the aberrant tRNA is accepted in the A-site, the second when it slips in the P-site). Another thing that may further increase the frequency of frameshifting is if the tRNA that is supposed to decode the A-site codon is slow to enter the A-site, which will provide a pause, during which the P-site peptidyl tRNA may shift frame (Farabaugh & Björk 1999; Urbonavicius et al. 2001). This model can explain frameshifting by tRNAs both with normal- sized and oversized anticodon loops. Based on the observation that efficient programmed +1 frameshifting in the TY3 retrotransposon occurred at a sequence where the P-site tRNA was not predicted to be able to bind the overlapping codon in the +1 frame (Vimaladithan & Farabaugh 1994), an

23 alternative model was proposed. In this model it was hypothesized that the first base of the A-site codon can sometimes be displaced from its normal orientation, which could allow the +1 codon to be presented in the A-site (Stahl et al. 2002). This would induce out of frame binding by an incoming aminoacyl-tRNA, similar to what was proposed in Curran and Yarus’ model above (Curran & Yarus 1987). As efficient frameshifting often requires a near-cognate peptidyl-tRNA in the P-site, this “flipping out” of the first A- site codon nucleotide would be more likely to occur in the presence of an unusual base pair (purine-purine clash or a pyrimidine-pyrimidine pair) at the wobble position of the P-site codon-anticodon complex (Stahl et al. 2002). This latter model can only explain +1 frameshifting, but experimentally it may be difficult to clearly distinguish between slippage of the peptidyl-tRNA or out of frame binding of the next tRNA, as the end result of both mechanisms would be the same. However, a mechanism that involves transient melting of the P-site codon-anticodon complex, followed by re- pairing to a different codon must exist to explain shifts larger than one nucleotide, such as +2 hopping, stop-hopping (+6) (O'Connor 2003) and the programmed 50 nucleotide translational bypass in phage T4 gene 60 (Herr et al. 2000).

A B C

EPA EPA EPA

Wild-type Defective near-cognate cognate Wild-type tRNA wins tRNA wins near-cognate tRNA Defective cognate tRNA enters slowly; EPA Pause EPA Defective cognate tRNA 3nt. 3nt. translocation translocation and pause and pause

+1 +1 +1

EPA EPA EPA

Figure 9. The dual error model. A Defective tRNA can induce frameshifting in three different ways: A; the defective tRNA is too slow in entering the A-site, allowing a near cognate tRNA to decode the A-site codon. After translocation to the P-site, the near cognate tRNA slips into an overlapping reading frame. B; the defective tRNA is too slow in entering the A-site, providing a pause when the P-site tRNA may slip. C; the defective tRNA decodes the codon in the A-site, but once it has been translocated into the P-site it can slip on the mRNA in either direction. Adapted from (Urbonavicius et al. 2001). ”Defective” in this model can mean either mutant or hypomodified.

25 1.5.4 The ribosomal P-site

As the codon-anticodon complex has already been tested in the A-site, there is no need to monitor the codon-anticodon complex in the P-site. It has been suggested that the stability of the codon-anticodon complex in the P-site is crucial for maintaining the correct reading frame (Hansen et al. 2003). Several areas of interaction between the P-site tRNA and both the 30S and 50S subunits can be seen in crystal structures (Korostelev & Noller 2007; Selmer et al. 2006; Yusupov et al. 2001). The anticodon stem is involved in interactions with parts of 16S rRNA as well as the carboxy termini of r- proteins S9 and S13 (Figure 10). The wobble base of tRNA is stacked on C1400 of 16S rRNA, and its ribose is stacked against G966. The D-stem interacts with helix 69 of 23S rRNA and the D- and TΨC-loops interact with residues of r-protein L5.

PTC L27 L16

G1338 S1 3 h69 A1229 C1230

A790 A1339

C A1340 o d o n S9 G966 C1400 Figure 10. Overview of tRNA-ribosome interactions in the P-site. tRNA is shown as cartoon representation. rRNA and protein residues with atoms within 3.8 Å of the tRNA is shown as sticks, and parts of the proteins are also shown as tubes. h69; helix 69 in 23S rRNA. PTC; peptidyl transferase center. 16S residues are numbered according to their location in E. coli 16S rRNA. The image was rendered with pymol (DeLano 2002), from the structure of the Thermus thermophilus 70S ribosome (Selmer et al. 2006), pdb codes 2j00 and 2j01.

27 2 RESULTS AND DISCUSSION

5 2.1 Synthesis of cmoP U34P in tRNA (Paper I)

Previously, mutants in aro genes, encoding enzymes in the biosynthetic 5 pathway leading to chorismic acid, were shown to lack cmoP U34P in their tRNA (Björk 1980; Hagervall et al. 1990). Chorismic acid is the last common precursor in the biosynthetic pathways for the three aromatic amino acids and the aromatic vitamins. In these studies, no biosynthetic intermediate except 5 the occasional presence of small amounts of hoP UP (5-hydroxy uridine) was seen (Björk 1980; Hagervall et al. 1990). Based on the knowledge that 5 Bacillus subtilis have moP U34P (5-methoxy uridine) in tRNA, and that one of 5 the two carbon atoms in cmoP UP originated from S-adenosyl-methionine (AdoMet), a biosynthetic pathway was proposed (Figure 11). Based on the following observations we set up a genetic screen in order to isolate mutants 5 of Salmonella enterica affected in the biosynthesis of cmoP U:P The lack of 5 cmoP U34P in an aro mutant leads to a decreased rate of frameshifting by

Pro tRNA cmo5UGG at CCC codons (Qian et al. 1998). A strain lacking

Pro tRNA GGG (ΔproL) and carrying the +1 frameshift mutation hisD3749 is able to grow on medium lacking histidine through suppression of the frameshift

Pro mutation when tRNA cmo5UGG reads a CCC codon in the hisD mRNA. Introduction of an aro mutation into such strain leads to loss of suppression - and thus the phenotype becomes HisP .P Of the many mutants tested in this screen, only one also showed the expected decrease in +1 frameshifting in a β-galactosidase assay. tRNA from this mutant did not have any detectable 5 cmoP U.P The transposon insertion responsible for the phenotype was found to

28 be in the gene yecO, in an operon also containing the gene yecP (we have re- named these genes cmoA and cmoB, respectively). Both cmoA and cmoB are predicted to encode AdoMet-dependent methyl transferases. By analyzing tRNA from different mutants of cmoA and cmoB we could see that any 5 5 mutant lacking cmoB (cmoB2 and cmoAB3) have hoP UP instead of cmoP U,P 5 5 while one cmoA (cmoA1) mutant had a mix of moP UP and hoP U.P Similarly, we 5 5 could also show that an aroD mutant also contain a mix of moP UP and hoP U,P although at much lower levels than in a cmoA1 mutant. When tRNA was 5 5 prepared from stationary phase cultures, the proportion of moP UP versus hoP UP increased both for a cmoA1 mutant and for an aroD mutant, but still no 5 cmoP UP was detected. Taken together, these results indicate that CmoB is 5 5 likely to be the methyl transferase responsible for producing moP UP from hoP UP (Figure 11).

5 Figure 11. Proposed pathway for biosynthesis of cmoP U34P (Hagervall et al. 1990) and Paper I. The grey arrows leading from chorismic acid to steps in the synthesis of 5 cmoP UP indicate a requirement for chorismic acid or an unknown derivative of it in those steps.

29 5 As cmoA and aroD mutants can produce moP UP only very slowly, the CmoB enzyme appears to require both CmoA and the presence of chorismic acid for full activity. As neither a cmoA mutant nor an aroD mutant produced any 5 5 cmoP U,P the same dependency apparently applies to the conversion of moP UP to 5 cmoP U.P It is difficult to imagine the chemical reaction required for 5 5 synthesizing cmoP UP from moP PU, but as only one of the carbon atoms in 5 cmoP UP originates from AdoMet it is likely that this carbon is the methyl 5 group from moP U.P As CmoA appears to be a methyl transferase as well, it is 5 unlikely to be the enzyme adding the carbon in the carboxyl group of cmoP U.P 5 One explanation to why cmoA mutants lack cmoP UP could be that several 5 enzymes in the biosynthetic pathway leading to the synthesis of cmoP UP and 5 its methyl ester (mcmoP U)P form a multi subunit complex, which is dependent on the presence of CmoA. Lack of CmoA could lead to disruption of this hypothetical complex, which could explain both the partial loss of CmoB 5 5 activity (hoP UP ÆmoP U)P and the complete loss of the activity of the enzyme(s) 5 5 required for the next step (moP UP ÆcmoP U).P The true role of CmoA in this 5 5 complex could be as the methyl transferase converting cmoP UP to mcmoP U.P 5 The nature of the link between the synthesis of cmoP UP and chorismic acid is still unknown, either chorismic acid itself or an unknown derivative of it is apparently important for at least three steps in the proposed pathway (grey arrows in Figure 11). Further biochemical and genetic analyses are needed to 5 find the missing genes required for the synthesis of cmoP UP and its methyl ester, and to understand the connection to the aro pathway.

30 5 2.2 Function of cmoP U34P in decoding (Paper I and II)

According to Crick’s wobble hypothesis, a Uridine at the wobble position of tRNA can pair with codons ending with A and G (Figure 6 and Table I) 5 (Crick 1966). cmoP U34P was predicted to expand the wobble capacity of uridine so that it in addition to pairing with A and G can pair with codons ending with U, but not C (Yokoyama et al. 1985). The biosynthetic 5 intermediate hoP U34P would not cause this expanded wobble capacity and would thus have the same base pairing properties as uridine. If these models are true, one may predict that a mutant lacking a G34-containing serine, proline, threonine, alanine, valine or leucine tRNA would not be viable, as such mutants would have no tRNA capable of reading the C-ending codons. 5 5 Furthermore the tRNAs of a cmoB mutant, having hoP UP instead of cmoP U,P should be poor at reading U-ending codons. The codon specificities of the 5 cmoP U-containingP leucine and serine tRNAs have been tested in in vitro translation systems, and these tRNAs behave as predicted from the models: they read their corresponding U-, A- and G-ending codons, but not the C- ending codons (Ishikura et al. 1971; Sørensen et al. 2005). In addition, the gene encoding the G34-containing leucine tRNA is essential in E. coli, 5 confirming the in vitro data by showing that the cmoP U-containingP leucine tRNA can not decode the CUC codon in vivo (Nishiyama & Tokuda 2005). Similarly, we have shown that the G34-containing threonine tRNA is essential (Paper II). Despite the above data that seems to support the models, mutants lacking the G34-containing alanine, valine and proline tRNAs are 5 viable (Paper I; Paper II; (Gabriel et al. 1996)), indicating that the cmoP U-P containing alanine, valine and proline tRNAs can read even the C-ending 5 codons. Interestingly, a strain with only the cmoP U-containingP proline tRNA

31 showed no reduction in growth rate unless it was combined with hypo- modification of the wobble nucleoside (Paper I). The reduction in growth rate was more obvious in a strain lacking the G34-containing tRNA than in a strain lacking the C34-containing tRNA, which was interpreted as a reduced

Pro function of tRNA cmo5UGG in reading mainly the U- or C-ending codons (Paper 5 I). A strain having only the cmoP U34P alanine tRNA had a reduced growth rate even when the tRNA was fully modified, and this phenotype was made very 5 much worse by hypo-modification. A strain with only the cmoP U-containingP valine tRNA showed a quite severe reduction in growth rate, which was further reduced by hypo-modification in a cmoB mutant.

5 Most of the in vitro translation data with tRNAs containing cmoP U34P have been done only with fully modified tRNA, sometimes in comparison to 5 totally unmodified tRNA, so the contribution of cmoP U34P to decoding properties have not been properly addressed (Ishikura et al. 1971; Kothe & Rodnina 2007; Mitra et al. 1979; Oda et al. 1969; Samuelsson et al. 1980; 5 Sørensen et al. 2005; Takai et al. 1999). To study the function of cmoP UP in decoding, we have used an in vivo A-site selection rate assay based on the programmed +1 frameshift site in the gene (prfB) encoding release factor 2 (RF2) (Curran & Yarus 1989) (Figure 12).

32 aminoacyl-tRNA enters A-site No β-gal.

Rt CUUXYZN lacZ (+1 frame) CUUXYZN lacZ (+1 frame) EPA EPA

p ep tid yl -tR NA s lip st o R +1 s fr am e

β-gal.

CUUXYZN lacZ (+1 frame) EPA

Figure 12. A-site selection rate assay. The lacZ gene is placed in the +1 frame after the programmed frameshift site from the prfB gene. A test codon (XYZ) is placed in place of the UGA stop codon present in the wild-type prfB site. At this site two competing reactions occur: When an aa-tRNA reads the test codon in the zero frame, translation will terminate at a stop codon following the site, and no betagalactosidase will be produced. Before the test codon is recognized, Peptidyl- Leu tRNAP P reading the P-site CUU codon may slip to the overlapping UUX codon in the +1 frame, leading to continued translation in the +1 frame and production of one molecule of β-galactosidase. As the rate of slippage (Rs) is assumed to be constant, the rate of aa-tRNA entry (Rt) to the test codon will be inversely proportional to the frameshift frequency (F). The rate can thus be expressed as Rt/Rs=(1/F)-1 (Curran & Yarus 1989).

The A-site selection rate determinations at proline (CCN), alanine (GCN) and 5 valine (GUN) codons in strains with only the cmoP U34-containingP tRNAs to read all four codons in the respective family boxes showed that the relative efficiencies of recognizing the U-, A- and G-ending codons was very similar, while recognition of the C-ending alanine and valine codons were much

33 lower. The rate of recognizing the C-ending proline codons was almost as high as the rates at the other proline codons, offering a possible explanation why this mutant did not have a growth rate reduction (Paper II). The expected results from the A-site selection rate assays in a cmoB mutant (if the wobble hypothesis and revised wobble rules were correct) would be a large reduction in the rate of recognizing the U-ending codons and perhaps the C-ending codons, while A- and G-ending codons should not be affected. The actual results were that the only changes that were consistent between the different family codon boxes were significant reductions in the recognition of G- ending codons. The reduction was largest for the alanine tRNA, which correlates with the fact that a mutant lacking the G34-containing alanine tRNA had the most severe synergistic phenotype when the remaining tRNA was hypo-modified. The data did not exclude effects on the U- or C-ending alanine or valine codons, as the assays for the cmoB mutant were done in the presence of the G34-containing alanine and valine tRNAs. However, in the mutant lacking the G34- and C34-containing proline tRNAs, hypo- modification caused only a small reduction in the rate of recognizing the CCU codon and a larger reduction in the rate of recognizing the G-ending codon. Based on these results, the revised wobble rules as well as the wobble hypothesis appear to need another revision. Recently published crystal Val 5 structures of ASLP P containing cmoP UP bound to the four valine codons (Weixlbaumer et al. 2007), offer an alternative molecular mechanism for the 5 function of cmoP UP that is better supported by our data (Figure 13). In all four 5 of these structures the ether oxygen of cmoP U34P is hydrogen bonded to the 2’-hydroxyl group of U33 of the ASL. The presence of this extra hydrogen 5 bond appears to “lock” cmoP UP in the correct position for forming a Watson-

34 5 Crick base pair. The ribose of cmoP U34P was in the 3’-endo conformation in all four structures, and not 2’-endo as suggested by Yokoyama et al for the 5 cmoP U-UP pair (Yokoyama et al. 1985). Neither Crick nor Yokoyama et al considered base pairs with only one hydrogen bond between the codon and 5 anticodon bases as stable enough, but in both of the cmoP U-pyrimidineP pairs only one hydrogen bond can be formed (Figure 13C and D) (Weixlbaumer et al. 2007).

A B

2’-OH O5 U33

cmo 5 U34 A(III) G(III) cmo 5 U34 C D

U(III) cmo 5 U34 C(III) cmo 5 U34 5 5 Figure 13. Structures of the four base pairs involving cmoP U34.P A: cmoP U-A.P B: 5 5 5 cmoP U-G.P C: cmoP U-U.P D: cmoP U-C.P The 2’-OH of uridine 33 and the ether oxygen 5 (O5) of cmoP UP are indicated with arrows in A. Hydrogen bonds are indicated by dashed lines. The images were rendered with PyMOL (DeLano 2002) from the crystal structures solved by Weixlbaumer et al (Weixlbaumer et al. 2007) (pdb codes 2UUC (A), 2UU9 (B), 2UUB (C) and 2UUA (D)).

35 5 The extra hydrogen bond between U33 and cmoP U34P compensates for the 5 5 “missing” hydrogen bond in the cmoP U-UP and cmoP U-CP pairs. As this extra 5 hydrogen bond is possible also with hoP U,P a cmoB mutant would not be expected to have any large effect on recognition of U- and C-ending codons, 5 consistent with our data (Paper II). The cmoP U-GP pair was in Watson-Crick geometry, which was made possible by stabilization of a usually very rare enol tautomer of uridine (Figure 13B and Figure 14) caused by the modification. A similar shift in the keto-enol equilibrium compared to uridine 5 5 can be seen for moP U,P but not hoP UP (Hillen et al. 1978). From these 5 5 observations one can predict that having hoP UP instead of cmoP UP would make recognition of G-ending codons less efficient, which is also consistent with our data (Paper II).

5 Figure 14. Keto-enol equilibrium of cmoP U.P

36 2.3 A dominant frameshift suppressor accumulates novel modified nucleosides in tRNA (Paper III)

The frameshift suppressor sufC mediates suppression of the +1 frameshift mutation hisC3737 (Riddle & Roth 1970). Later characterization of sufC mutants has shown that they contain two mutations (sufX and sufY), both contributing to frameshift suppression. sufX (proL) was shown to encode

Pro mutant tRNA GGG , while the sufY mutation has not been previously examined (Sroga et al. 1992). To further characterize the sufY mutation we examined which frameshift mutations it could suppress and found that the minimal sequence at which it frameshifts is CCC-CAA (Pro-Gln) (Paper III). N- terminal sequence analysis of a peptide resulting from frameshifting at CCC- CAA-AGC-UAA (encoding Pro-Gln-Ser-Stop) was consistent with a proline tRNA slipping at the CCC codon, placing a lysine (AAA) codon in the A-site. This could mean that the mutation affects either a proline tRNA or a glutamine tRNA, but as sufY did not induce frameshifting at other sites containing proline codons, it was more likely to be a defective glutamine tRNA that caused frameshifting. This was confirmed as the level of

Gln aminoacylation of tRNA cmnm5s2UUG was reduced. Genetic mapping of sufY revealed that the mutation responsible for the phenotype was located in the gene ybbB and was dominant over the wild-type allele and had a “gain of function” phenotype. Bulk tRNA hydrolysates from a sufY mutant contained two unknown dinucleotides (UK1 and UK2), and one of these was also found

Gln in hydrolysates of purified tRNA cmnm5s2UUG . Mass spectrometry (MS) analysis of purified UK2 identified this compound as the dinucleotide U*pU, where 5 2 U* is an unknown derivative of the wobble nucleoside mnmP sP P UP containing

37 an extra C10B HB 17B -B hydrocarbon substituent attached to the base. The exact

structure of this C10B HB 17B -unitB could not be determined, but the data is consistent with it being a geranyl group. Mutants affecting different steps of 5 the synthesis of the (c)mnmP -P sidechain of the wobble modification accumulated different unknown compounds (UK1, UK2 or UK3, derivatives 5 2 5 2 5 2 of cmnmP sP P U,P mnmP sP P PU and nmP sP P U,P respectively), while a mutant (mnmA) unable to introduce the 2-thiol group of the modification did not contain any detectable unknown compound. Together these results showed that the extra 5 C10B HB 10B -B group was not attached to the (c)mnmP -sidechain,P but most likely on the 2-thiol group (Figure 15). O R NH N

C10H17 N S HO O

OH OH Figure 15. Hypothetical structure of the 5’-component of the dinucleotides UK1, UK2 and UK3. R: -CH3B B (UK2); -CH2B COOHB (UK1); -H (UK3). The structure of the –C10B HB 17B B chain is unknown.

As wild-type Salmonella contained a very low, but detectable, level of UK2 in bulk tRNA, the sufY mutation enhanced a reaction the wild-type YbbB protein was already capable of. YbbB has been shown to catalyze the 5 2 5 2 conversion of mnmP sP P UP into mnmP seP P UP (exchanging the sulphur for selenium) in tRNA (Wolfe et al. 2004). As the UK’s contained sulfur and not

38 selenium, and a selD mutant, lacking the selenium donor selenophosphate, still made UK1 and UK2, the selenation reaction is not part of the synthesis of the UK’s.

Gln As the wobble nucleoside of tRNAP P interacts with the anticodon binding

domain of GlnRS (Rould et al. 1991), the presence of the bulky C10B HB 17B -B side

Gln chain in tRNA U*UG in a sufY mutant is expected to cause a decrease in aminoacylation, as we observed. It is also likely that the abnormal structure on the wobble nucleoside prevents the tRNA from entering the ribosomal A- site. As frameshifting by a sufY mutant was also inhibited by an aroD mutation, which causes hypo-modification of the wobble nucleoside in

Pro tRNA cmo5UGG , the likely cause of frameshifting can be explained by the dual error model (Figure 9 and Paper III, supplementary figure 1): The first error is

Pro when tRNA cmo5UGG wins the competition for the CCC codon in the A-site. After peptidyl transfer and a normal three nucleotide translocation, this peptidyl-tRNA is prone to slip into the overlapping CCC codon in the +1 frame. The second error is the pause that results from slow entry of the poorly

Gln aminoacylated and structurally abnormal tRNA U*UG into the A-site, which gives the peptidyl-tRNA more time during which it can shift frame. In the

Pro original sufC mutants, the mutant tRNA GGG enhanced frameshifting further

Pro by allowing tRNA cmo5UGG to read the CCC codon more often.

39 2.4 The “ribosomal grip” of the peptidyl-tRNA is critical for reading frame maintenance? (Paper IV)

Some of the earliest characterized bacterial frameshift suppressors (sufA, sufB, sufD, sufJ) had insertions in the anticodon loops of tRNAs, and induced frameshifts at sites that were consistent with a model where the affected tRNAs had a functional four base anticodon. According to the model, these mutant tRNAs would recognize a four base codon in the ribosomal A-site, followed by a quadruplet translocation into the P-site (Bossi & Smith 1984; Roth 1981). There are frameshift suppressors that can not be explained by a

Pro four base anticodon. Many mutations in the gene (proL) encoding tRNA GGG (including the classical sufB2 mutant) have been found to induce +1 frameshifting at CCC (Pro) codons, but the error occurs when a proline tRNA occupies the P-site and the A-site is empty (Qian & Björk 1997). In addition,

Pro it is not the mutant tRNA itself, but rather the wild-type tRNA cmo5UGG that is responsible for frameshifting (even in the sufB2 mutant, despite its apparent four base anticodon) (Qian et al. 1998). Similarly, a deletion of the gene

Pro (proL) encoding tRNA GGG (Paper I) or over-expression of the gene (proM)

Pro encoding tRNA cmo5UGG also induce frameshifting (O'Connor 2002) at CCC codons.

To gain better understanding of the mechanism of frameshifting by

Pro tRNA cmo5UGG and the mechanism of reading frame maintenance in general we

Pro performed a selection for mutants in the gene proM (encoding tRNA cmo5UGG ) capable of suppressing two different hisD +1 frameshift mutations, which

40 both contain a CCC (Pro) codon in their frameshift windows (Paper IV). + Characterization of 108 independently isolated HisP P proM mutants resulted in identification of eleven different alterations of ten different positions in

Pro tRNA cmo5UGG . As all of these suppressors increase frameshifting both in the

Pro absence and presence of tRNA GGG , which is the true cognate tRNA for CCC codons, the mutant tRNAs are themselves directly responsible for

Pro frameshifting. Frameshifting by all tRNA cmo5UGG variants is inhibited by expression of more of the tRNA that should read the next in-frame codon,

Pro which is consistent with slippage of peptidyl- tRNA cmo5UGG in the P-site following a normal three nucleotide translocation, but is not consistent with a

Pro four nucleotide translocation or out-of-frame binding by tRNA cmo5UGG . Furthermore, some of the mutations caused dramatic reductions in growth

Pro rate when combined with absence of tRNA GGG (which reads CCG codons)

Pro and mild reductions in growth rate when combined with lack of tRNAGGG (which reads CCC and CCU codons). These synergistic phenotopes were partially relieved by over-expressing prolyl-tRNA synthase (ProRS),

Pro indicating that the mutant tRNA cmo5UGG are poorly aminoacylated. As ProRS did not inhibit frameshifting, the frameshifting is not caused by poor

Pro aminoacylation, but more likely by poor function of peptidyl- tRNA cmo5UGG in the P-site. Several of the mutations affect parts of the tRNA molecule that are very close to residues of the ribosomal P-site in crystal structures. We suggest that these mutations reduce the strength of the interactions between peptidyl-

Pro tRNA cmo5UGG and residues in the ribosomal P-site. Consequently, the ribosome may loose its grip of the peptidyl-tRNA and its codon-anticodon

41 complex, leading to an increased risk for frameshifting. We hypothesize that the ribomal P-site has a leading role in preventing frameshifting, by keeping a tight grip of peptidyl-tRNA.

42 3 CONCLUSIONS

1. The predicted methyl transferases CmoA and CmoB are required for 5 the synthesis of cmoP UP at the wobble position of transfer RNA (Paper I).

5 2. Proline, alanine and valine tRNAs containing cmoP U34P can read all 5 four of their respective codons. A threonine tRNA containing cmoP U34P can not read all four threonine codons (Paper I and II).

5 3. cmoP U34P is important for efficient reading of G-ending proline, alanine and valine codons (Paper II).

4. The frameshift suppressor sufY leads to accumulation of novel bulky Gln nucleosides at the wobble position of tRNAP P. This leads to a deficiency Gln for glutaminyl-tRNAP ,P causing frameshifting by wild-type peptidyl-

Pro tRNAcmo5UGG at CCC-CAA (Pro-Gln) (Paper III).

Pro 5. Mutant variants of tRNAcmo5UGG induce frameshifts in the P-site. Several of the mutations affect residues involved in interactions with ribosomal P-site residues, suggesting an important role of the residues making up the ribosomal P-site in maintaining a firm grip of peptidyl- tRNA to prevent frameshifting (Paper IV).

43 ACKNOWLEDGEMENTS

First and most of all I wish to thank my supervisor, Glenn Björk. Your never-ending optimism, patience and curiosity have been great sources of inspiration and support for me during my time as a Ph. D student.

I’m also very grateful to our excellent technicians; Gunilla, Kristina and Kerstin. Thank you for all your help in the lab, for always helping me find solutions, chemicals and protocols. I’ll add an extra “thank you” to Gunilla for doing all β-gal assays for paper II, while I was doing all the fun parts (Salmonella genetics). Thanks to past members of group GB: Peng, Jaunius, Hans, Ramune and Jerome, who have all contributed to making a very good group to work in. Thanks to the rest of the people in our journal clubs and group meetings for many interesting discussions; Tord, past and present members of group AB: Anders, Anders, Chen, Jian, Marcus and Bo, and group MW: Micke and Stefan. I want to acknowledge some other guys that I have shared the lab, many lunches, crazy discussions and lots of whisky with during the first few years at the department: Mattias and Jörgen. And since I mentioned whisky; thanks to the rest of the old whisky club. The department would of course be nothing without the valuable services provided by the secretaries, Johnny, the media- and dishwashing staff and the Beer Corner. Last, but not least, I thank my family: My parents Kjell and Britt-Mari, my brother Daniel and sister Sara for their support, and my sisters children Alexander (“du”) and Felicia (“ia”) for being who they are.

44 REFERENCES

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