DOCSLIB.ORG

A Mutant T7 RNA Polymerase As a DNA Polymerase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Mutant T7 RNA Polymerase As a DNA Polymerase The EMBO Journal vol.14 no.18 pp.4609-4621, 1995 A mutant T7 RNA polymerase as a DNA polymerase Rui Sousa1 and Robert Padilla in dNTP Km (Ricchetti and Buc, 1993) and T7 DNA- directed RNA polymerase (RNAP) can also use RNA as Department of Biochemistry, University of Texas Health Science a template (Konarska and Sharp, 1989). These are not Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, exceptional observations, since it is a general property of TX 78212, USA polymerases that they display relaxed template specificity, 'Corresponding author at least in vitro. While template specificity may be relaxed, polymerase substrate specificity is normally extremely We have identified a T7 RNA polymerase (RNAP) stringent. T7 DNAP, for example, displays at least 2000- mutant that efficiently utilizes deoxyribonucleoside tri- fold selectivity for dNTPs over rNTPs, even in Mn2+ phosphates. In vitro this mutant will synthesize RNA, buffer, which relaxes the ability of the polymerase to DNA or 'transcripts' of mixed dNMP/rNMP composi- discriminate between dNTPs and ddNTPs (Tabor and tion depending on the mix of NTPs present in the Richardson, 1989). The structural determinants of such synthesis reaction. The mutation is conservative, stringent specificity remain undefined. changes Tyr639 within the active site to phenylalanine We report here the identification of mutant T7 RNAPs and does not affect promoter specificity or overall that display the ability to use dNTPs. The mutations occur activity. Non-conservative mutations of this tyrosine in Tyr639 within motif B (Delarue et al., 1990) of T7 also reduce discrimination between deoxyribo- and RNAP. Two observations impelled us to examine the ribonucleoside triphosphates, but these mutations also substrate discrimination and miscoding properties of these cause large activity reductions. Of 26 mutations of mutants. It had been found that mutations in the corres- other residues in and around the active site examined ponding conserved tyrosine in DNAP I increased mis- none showed marked effects on rNTP/dNTP dis- coding (Polesky et al., 1990; Carrol et al., 1991). It was crimination. Mutations of the corresponding tyrosine also found that transcripts synthesized by a T7 RNAP in DNA polymerase (DNAP) I increase miscoding, Y639F mutant in vivo yielded 33-50% of the protein per though effects on dNTP/rNTP discrimination for the transcript compared with transcripts synthesized by the DNAP I mutations have not been reported. This con- wild-type enzyme (Makarova et al., 1995). The latter served tyrosine may therefore play a similar role in phenotype was unique to the Y639F mutant amongst a many polymerases by sensing incorrect geometry in number of other active site mutants examined for in vivo the structure of the substrate/template/product due to expression and indicated that Y639F transcripts contained inappropriate substrate structure or mismatches. T7 a defect that led to their being inefficiently translated. RNAP can use RNA templates as well as DNA templates These observations implied that mutations in Tyr639 and is capable of both primer extension and de novo might cause increased misincorporation, either increased initiation. The Y639F mutant retains the ability to use mismatch synthesis (miscoding) or incorporation of sub- RNA or DNA templates. Thus this mutant can display strates of inappropriate structure. We have therefore de novo initiated or primed DNA-directed DNA characterized the ability of the Y639 mutants, as well as polymerase, reverse transcriptase, RNA-directed a large number of other active site mutants, to miscode RNA polymerase or DNA-directed RNA polymerase or to use dNTPs in both Mg2+ and Mn2+ buffers. Our activities depending simply on the templates and sub- results point to a specialized role for Tyr639 in T7 RNAP strates presented to it in the synthesis reaction. (and the corresponding tyrosine in other polymerases) in Keywords: mutagenesis/RNA polymerase/substrate speci- ensuring that substrates to be added to the growing nucleic ficity/transcription acid have the correct structure. They reveal that both transcript and substrate structure affect the efficiency with which the transcript is extended. They show that the Introduction restriction of unprimed initiation to RNAPs is not due to an intrinsic property of ribo- versus deoxyribonucleotides, One classification of nucleic acid polymerases relies on but simply to the selectivity of the polymerase active site. their different template specificities (RNA or DNA), They also present researchers with a novel reagent that substrate specificities (rNTPs or dNTPs) and mode of expands the structural range of nucleic acids that can be initiation (de novo or primed). These designations usually enzymatically synthesized in vitro. refer to the template and substrate specificities displayed in vivo during the fulfillment of a polymerase's biological Results function. In vitro, polymerases can display novel activities, albeit with reduced efficiency and/or under non-physio- Structure of the transcripts synthesized by Y639F logical conditions. Escherichia coli DNA-directed DNA and the wild-type enzyme with rNTPs and dNTPS polymerase (DNAP) I, for example, can use RNA as a Figure 1 shows transcription reactions carried out with template, though with a concomitant -100-fold increase the wild-type enzyme or the Y639F mutant polymerase K Oxford University Press 4609 R.Sousa and R.Padilla A BIC FIG HTI J IK N|O R|S T|U X geneous sequence abortive transcripts or 59 base run-off DIEI I I LIM PIQ VIW Pol: MWTMu WT Mu WT M.lu WT M. WT M.u WT M. WTM.lWTuWT Mu WT MIu WT Mu WTM.u products made and instead long poly(G) transcripts, as in rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP rGTP are NTPs: rATP dATP rATP rATP rATP rATP rATP rATP dATP dATP lane A, made. Adding dATP to reactions lacking rATP rCTP rCTP rCTP dCTP rCTP rCTP rCTP rCTP dCTP dCTP I rUTP rUTP rUTP rUTP rUTPI dTTP dTTP rUTP dTTP does not change the transcripts produced by the wild-type Rutlo - M enzyme (lane G). However, with the mutant enzyme we observe that addition of dATP (lane H) allows synthesis of a long run-off transcript, as well as synthesis of heterogeneous sequence abortive transcripts that do not co-migrate with the poly(G) transcripts. Observation of an abortive transcript in lane H running near the position of the 4H band in lane D confirms extension of the GGG trimer with an A, but note that the major 4mer transcript in lane H migrates close to, but not precisely with, the major 4mer in lane D or in adjacent lane I. This is consistent with the expectation that these 4mers will have identical sequence and length, but different structure (i.e. ._a rGrGrGrA in lanes D and I, rGrGrGdA in lane H). It should also be noted that some poly(G) transcript .7 ._w synthesis is observed in lane H. For example, in lane H we observe both a heterogeneous sequence 4mer migrating near the _=11111o ..:. 4H position and a smaller amount of 4mer band migrating ,LH 04 :-W 4 at the _w 4G position. When four rNTPs are present (lane C or D) synthesis of poly(G) transcripts is more completely suppressed. This indicates that dATP is utilized by Y639F, X - but not as efficiently as rATP. -,- Ck When rCTP is omitted from the reaction, transcripts .. terminate predominately at the 6mer length, because rCMP is normally first incorporated at position 7 (lanes I and J). Fig. 1. Structure of transcription products produced by Y639F and Addition of dCTP does not allow extension of the wild-type T7 RNAP in the presence of various combinations of rNTPs 6mer and dNTPs. The template was pT75 (Tabor and Richardson, 1985) cut in reactions with the wild-type enzyme (lane K). However, with HindIH so that transcription from its T7 promoter generated a 59 addition ofdCTP to reactions with Y639F allows extension base run-off transcript. Electrophoresis was on a 20% polyacrylamide- beyond the 6mer length and synthesis of the run-off 6 M urea gel. Plasmid and polymerases were at concentrations of transcript (lane L). Again, the following should be noted: l0e M and NTP concentrations were 0.5 mM (all rNTPs and dTTP), (i) transcripts larger than 6 bases do not 1 mM (dATP, dGTP) or 5 mM (dCTP). [,y-32P]GTP was added to co-migrate with radiolabel the transcription products. Wild-type (WT) or Y639F mutant their counterparts in lane C or D, consistent with the (Mu) polymerases and NTPs used are as indicated. Poly(rG) products expected structural difference despite length and sequence of various sizes are labeled in lane a (2G, 3G, etc.) and heterogeneous identity; (ii) there is more termination at the 6- and 7mer sequence abortive transcripts of different lengths are indicated by 4H, points in lane L than in lane C or D, that 5H, etc. in lane C. Lanes Q-T are a 10-fold longer exposure of lanes indicating Y639F M-P. uses dCTP well, but not as efficiently as it utilizes rCTP. In lanes M and N UTP was omitted from the reactions. Lanes Q-T show a 10-fold longer exposure of lanes M- and a T7 d10 promoter template. Transcription by T7 P. Within the set of four NTPs UTP is unique on this RNAP, like other RNAPs, is characterized by an initial, template, since it first becomes incorporated into the poorly processive 'abortive' phase of transcription during transcript at the 13 base position. This corresponds to a which the short, nascent transcript frequently dissociates transcript length subsequent to the transition from abortive from the ternary complex. When the transcript reaches a to processive transcription. As a consequence of this length of ;-9 bases transcription becomes highly processive transition the ternary complex becomes more stable and the transcript becomes stably associated with the (Martin et al., 1988).
Load more

Recommended publications
  • Biomolecules
    CHAPTER 3 Biomolecules 3.1 Carbohydrates In the previous chapter you have learnt about the cell and 3.2 Fatty Acids and its organelles. Each organelle has distinct structure and Lipids therefore performs different function. For example, cell membrane is made up of lipids and proteins. Cell wall is 3.3 Amino Acids made up of carbohydrates. Chromosomes are made up of 3.4 Protein Structure protein and nucleic acid, i.e., DNA and ribosomes are made 3.5 Nucleic Acids up of protein and nucleic acids, i.e., RNA. These ingredients of cellular organelles are also called macromolecules or biomolecules. There are four major types of biomolecules— carbohydrates, proteins, lipids and nucleic acids. Apart from being structural entities of the cell, these biomolecules play important functions in cellular processes. In this chapter you will study the structure and functions of these biomolecules. 3.1 CARBOHYDRATES Carbohydrates are one of the most abundant classes of biomolecules in nature and found widely distributed in all life forms. Chemically, they are aldehyde and ketone derivatives of the polyhydric alcohols. Major role of carbohydrates in living organisms is to function as a primary source of energy. These molecules also serve as energy stores, 2021-22 Chapter 3 Carbohydrade Final 30.018.2018.indd 50 11/14/2019 10:11:16 AM 51 BIOMOLECULES metabolic intermediates, and one of the major components of bacterial and plant cell wall. Also, these are part of DNA and RNA, which you will study later in this chapter. The cell walls of bacteria and plants are made up of polymers of carbohydrates.
    [Show full text]
  • A Simple Colorimetric RNA Polymerase Assay
    Virology 274, 429–437 (2000) doi:10.1006/viro.2000.0492, available online at http://www.idealibrary.com on Exploiting Polymerase Promiscuity: A Simple Colorimetric RNA Polymerase Assay William Vassiliou,* Jeffery B. Epp,† Bin-Bin Wang,* Alfred M. Del Vecchio,‡,1 Theodore Widlanski,§ and C. Cheng Kao*,2 *Department of Biology, Indiana University, Bloomington, Indiana 47405; †Dow AgroSciences, Indianapolis, Indiana 46268; ‡SmithKline Beecham Inc., Collegeville, Pennsylvania 19426; and §Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Received May 18, 2000; accepted June 29, 2000 We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the ␥-phosphate were synthesized (PNP–NTPs). These PNP–NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophe- nylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP–NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis. © 2000 Academic Press INTRODUCTION polymerases are primarily tested using radioactive assays that require special handling of the isotope RNA and DNA polymerases carry out the synthesis of and are incompatible with continuous monitoring of oligonucleotides by transfer of a nucleoside monophos- polymerase activity (Ferrari et al., 1999).
    [Show full text]
  • Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences
    molecules Review Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences Yaroslav Staroseletz 1,†, Svetlana Gaponova 1,†, Olga Patutina 1, Elena Bichenkova 2 , Bahareh Amirloo 2, Thomas Heyman 2, Daria Chiglintseva 1 and Marina Zenkova 1,* 1 Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev’s Ave. 8, 630090 Novosibirsk, Russia; [email protected] (Y.S.); [email protected] (S.G.); [email protected] (O.P.); [email protected] (D.C.) 2 School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Rd., Manchester M13 9PT, UK; [email protected] (E.B.); [email protected] (B.A.); [email protected] (T.H.) * Correspondence: [email protected]; Tel.: +7-383-363-51-60 † These authors contributed equally to this work. Abstract: RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleav- age, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes Citation: Staroseletz, Y.; Gaponova, and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonu- S.; Patutina, O.; Bichenkova, E.; cleotide recognition motifs (or their structural
    [Show full text]
  • Mirna Research Guide Mirna Guide Cover Final.Qxd 9/28/05 10:24 AM Page 4
    miRNA_guide_cover_final.qxd 9/28/05 10:24 AM Page 3 miRNA Research Guide miRNA_guide_cover_final.qxd 9/28/05 10:24 AM Page 4 Contents Introduction to microRNAs and Experimental Overview Introduction to microRNAs . .1 miRNA Experimental Overview . .2 microRNA Isolation and Enrichment miRNA Isolation . .3 mirVana™ miRNA Isolation Kits . .3 “miRNA Certified” FirstChoice® Total RNA . .3 RecoverAll™ Total Nucleic Acid Isolation Kit . .3 miRNA Enrichment . .4 flashPAGE™ Fractionator System . .4 Global microRNA Expression Profiling miRNA Expression Profiling . .5 Overview of the mirVana™ Array System . .6 mirVana™ miRNA Labeling Kit . .7 mirVana™ miRNA Probe Set . .7 mirVana™ miRNA Bioarrays . .8 Detection and Quantification of Specific microRNAs mirVana™ miRNA Detection Kit . .9 mirVana™ miRNA Probe and Market Kit . .9 mirVana™ miRNA Probe Construction Kit . .10 mirVana™ qRT-PCR miRNA Detection Kit . .11 microRNA Functional Analysis miRNA Functional Analysis . .12 Anti-miR™ miRNA Inhibitors . .12 Pre-miR™ miRNA Precursor Molecules . .13 siPORT™ NeoFX™ Transfection Agent . .13 pMIR-REPORT™ miRNA Expression Reporter Vector . .13 microRNA Information Resources miRNA Resource . .14 miRNA Database . .14 miRNA Array Resource . .14 Introduction to microRNAs . .14 miRNA Application Guide . .15 Technical miRNA Seminars . .15 Highly Trained miRNA Technical Support Scientists . .15 miRNA e-Updates . .15 TechNotes Newsletter . .15 Reference Relative miRNA Expression Among Common Cell Types . .16 miRNA_guide_guts_final.qxd 9/28/05 10:26 AM Page 1 Introduction to microRNAs and Experimental Overview Introduction to microRNAs Overview of microRNA processing Small regulators with global impact miRNAs are transcribed as regions of longer RNA molecules that can be as long as 1000 nt (Figure 3). Description of microRNAs MicroRNAs (miRNAs) are evolutionarily conserved, small, noncoding RNA mol- The longer RNA molecules are processed in the nucleus into hairpin RNAs of ecules that regulate gene expression at the level of translation (Figure 1).
    [Show full text]
  • The Conserved Structure of Plant Telomerase RNA Provides the Missing Link for an Evolutionary Pathway from Ciliates to Humans
    The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans Jiarui Songa, Dhenugen Logeswaranb,1, Claudia Castillo-Gonzáleza,1, Yang Lib, Sreyashree Bosea, Behailu Birhanu Aklilua, Zeyang Mac,d, Alexander Polkhovskiya,e, Julian J.-L. Chenb,2, and Dorothy E. Shippena,2 aDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843; bSchool of Molecular Sciences, Arizona State University, Tempe, AZ 85287; cNational Maize Improvement Center of China, China Agricultural University, 100193 Beijing, China; dCollege of Agronomy and Biotechnology, China Agricultural University, 100193 Beijing, China; and eCenter of Life Sciences, Skolkovo Institute of Science and Technology, 121205 Moscow, Russian Federation Edited by Thomas R. Cech, University of Colorado Boulder, Boulder, CO, and approved October 24, 2019 (received for review September 4, 2019) Telomerase is essential for maintaining telomere integrity. Although transcribed by RNA polymerase III (Pol III) (6, 7). The La-related telomerase function is widely conserved, the integral telomerase protein P65 in Tetrahymena recognizes the 3′ poly-U tail of TR RNA (TR) that provides a template for telomeric DNA synthesis has and bends the RNA to facilitate telomerase RNP assembly (8, 9). diverged dramatically. Nevertheless, TR molecules retain 2 highly In contrast, fungi maintain much larger TR molecules (900 to conserved structural domains critical for catalysis: a template- 2,400 nt) that are transcribed by RNA polymerase II (Pol II) (3). proximal pseudoknot (PK) structure and a downstream stem-loop The 3′ end maturation of fungal TRs requires components of the structure. Here we introduce the authentic TR from the plant canonical snRNA biogenesis pathway and results in RNP assembly Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT.
    [Show full text]
  • RNA-Dependent RNA Polymerase Speed and Fidelity Are Not the Only Determinants of the Mechanism Or Efficiency of Recombination
    G C A T T A C G G C A T genes Article RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination Hyejeong Kim y, Victor D. Ellis III, Andrew Woodman, Yan Zhao, Jamie J. Arnold y and , Craig E. Cameron y * Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA; [email protected] (H.K.); [email protected] (V.D.E.); [email protected] (A.W.); [email protected] (Y.Z.); [email protected] (J.J.A.) * Correspondence: [email protected]; Tel.: +1-919-966-9699 Present address: Department of Microbiology and Immunology, School of Medicine, University of North y Carolina at Chapel Hill, 125 Mason Farm Rd., Chapel Hill, NC 27599-7290, USA. Received: 15 October 2019; Accepted: 21 November 2019; Published: 25 November 2019 Abstract: Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination.
    [Show full text]
  • T7 RNA Polymerase from Escherichia Coli BL 21/Par 1219 Nucleoside-Triphosphate: RNA Nucleotidyltransferase (DNA-Directed), EC 2.7.7.6 Cat
    For life science research only. Not for use in diagnostic procedures. T7 RNA Polymerase From Escherichia coli BL 21/pAR 1219 Nucleoside-triphosphate: RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6 Cat. No. 10 881 767 001 y Version 22 1,000 U Content version: March 2016 Cat. No. 10 881 775 001 5,000 U Store at Ϫ15 to Ϫ25° C Product Overview Standard Transcription Assay Pack Content Additional Reagents Required • lin. template DNA including T7 RNA promotor Vial Content • Ribonucleoside triphosphates T7 RNA • 1,000 U • labeled nucleotide (according to application) Polymerase • 5,000 U •RNAse inhibitor Enzyme storage buffer: 10 mM Potassium phosphate, • Water, PCR Grade 200 mM KCl, 0.1 mM EDTA, 30 mM 2-mercaptoethanol, 50% glycerol (v/v), 0.1% Tween 20, pH 7.9 (+4°C). Radioactive Assay Supplied Transcrip- Buffer composition (10x conc.): Pipet the following components into a microfuge tube, mix and make up to a tion buffer 0.4 M Tris-HCl, pH 8.0 (+20°C), 60 mM MgCl2 , 100 mM final volume of 20 µl: dithiothreitol, 20 mM spermidine. Storage and Stability Reagent Volume/Concentration Stable at -15 to -25°C until the expiration date printed on the label. Template DN A 0.5 µg Product Description Nucleotides ATP, GTP, CTP, UTP each 0.5 mM final T7 RNA polymerase is commonly used to transcribe DNA which has been α 32 cloned into vectors which have two phage promoters in opposite orientation. Labeled nucleotide [ - P] CTP [400 Ci/ 0.1 µl aqueous solution RNA can be selectively synthesized from either strand of the insert DNA with mmol; 15 TBq/mmol] different polymerases.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Riboprobe(R) in Vitro Transcription Systems Technical Manual TM016
    TECHNICAL MANUAL Riboprobe® in vitro Transcription Systems InstrucƟ ons for use of Products P1420, P1430, P1440, P1450 and P1460 Revised 10/13 TM016 tm016.1013:EIVD_TM.qxd 9/26/2013 11:10 AM Page 1 Riboprobe® in vitro Transcription Systems All technical literature is available on the Internet at www.promega.com/protocols Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail [email protected] 1. Description..........................................................................................................1 2. Product Components.........................................................................................3 3. General Considerations....................................................................................4 A. Properties of Promega Vectors Suitable for in vitro Transcription ..............4 B. Applications of Promega Vectors ......................................................................6 C. General Cloning Techniques ..............................................................................6 4. RNA Transcription in vitro .............................................................................7 A. DNA Template Preparation................................................................................7 B. Synthesis of High-Specific-Activity Radiolabeled RNA Probes ...................8 C. Determining Percent Incorporation and Probe Specific Activity ...............10
    [Show full text]
  • Mutation of a DNA Polymerase Into an Efficient RNA Polymerase
    Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase Gang Xia, Liangjing Chen, Takashi Sera, Ming Fa, Peter G. Schultz*, and Floyd E. Romesberg* Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved March 22, 2002 (received for review October 30, 2001) The creation of novel enzymatic function is of great interest, but of an attached substrate. However, the substrate was attached to remains a challenge because of the large sequence space of the major phage coat protein, pVIII, raising the concern of proteins. We have developed an activity-based selection method to cross-reactivity between a polymerase on one phage and a evolve DNA polymerases with RNA polymerase activity. The Stoffel substrate attached to another phage. Cross-reactivity will com- fragment (SF) of Thermus aquaticus DNA polymerase I is displayed promise the association of genotype with phenotype and inhibit on a filamentous phage by fusing it to a pIII coat protein, and the the successful evolution of desired function (see below). substrate DNA template͞primer duplexes are attached to other We have developed an activity-based selection method, in which adjacent pIII coat proteins. Phage particles displaying SF poly- a DNA polymerase and its substrate are both attached to the minor merases, which are able to extend the attached oligonucleotide phage coat protein, pIII. The pIII proteins are localized to only one primer by incorporating ribonucleoside triphosphates and biotin- end of the phage particle (Fig.
    [Show full text]
  • Nucleosides & Nucleotides
    Nucleosides & Nucleotides Biochemistry Fundamentals > Genetic Information > Genetic Information NUCLEOSIDE AND NUCLEOTIDES SUMMARY NUCLEOSIDES  • Comprise a sugar and a base NUCLEOTIDES  • Phosphorylated nucleosides (at least one phosphorus group) • Link in chains to form polymers called nucleic acids (i.e. DNA and RNA) N-BETA-GLYCOSIDIC BOND  • Links nitrogenous base to sugar in nucleotides and nucleosides • Purines: C1 of sugar bonds with N9 of base • Pyrimidines: C1 of sugar bonds with N1 of base PHOSPHOESTER BOND • Links C3 or C5 hydroxyl group of sugar to phosphate NITROGENOUS BASES  • Adenine • Guanine • Cytosine • Thymine (DNA) 1 / 8 • Uracil (RNA) NUCLEOSIDES • =sugar + base • Adenosine • Guanosine • Cytidine • Thymidine • Uridine NUCLEOTIDE MONOPHOSPHATES – ADD SUFFIX 'SYLATE' • = nucleoside + 1 phosphate group • Adenylate • Guanylate • Cytidylate • Thymidylate • Uridylate Add prefix 'deoxy' when the ribose is a deoxyribose: lacks a hydroxyl group at C2. • Thymine only exists in DNA (deoxy prefix unnecessary for this reason) • Uracil only exists in RNA NUCLEIC ACIDS (DNA AND RNA)  • Phosphodiester bonds: a phosphate group attached to C5 of one sugar bonds with - OH group on C3 of next sugar • Nucleotide monomers of nucleic acids exist as triphosphates • Nucleotide polymers (i.e. nucleic acids) are monophosphates • 5' end is free phosphate group attached to C5 • 3' end is free -OH group attached to C3 2 / 8 FULL-LENGTH TEXT • Here we will learn about learn about nucleoside and nucleotide structure, and how they create the backbones of nucleic acids (DNA and RNA). • Start a table, so we can address key features of nucleosides and nucleotides. • Denote that nucleosides comprise a sugar and a base.
    [Show full text]
  • Sequential Structures Provide Insights Into the Fidelity of RNA Replication
    Sequential structures provide insights into the fidelity of RNA replication Cristina Ferrer-Orta*, Armando Arias†, Rosa Pe´ rez-Luque*, Cristina Escarmi´s†, Esteban Domingo†, and Nuria Verdaguer*‡ *Institut de Biologia Molecular de Barcelona, Parc Cientı´ficde Barcelona, Josep Samitier 1-5, E-08028 Barcelona, Spain; and †Centro de Biologı´aMolecular ‘‘Severo Ochoa,’’ Cantoblanco, E-28049 Madrid, Spain Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved April 16, 2007 (received for review February 6, 2007) RNA virus replication is an error-prone event caused by the low evidence of how the physiological substrates bind the large fidelity of viral RNA-dependent RNA polymerases. Replication exposed active site of the picornavirus RDRPs (12, 13). In fidelity can be decreased further by the use of mutagenic ribonu- addition, the structure of the complex between the polymerase cleoside analogs to a point where viral genetic information can no 3D and its protein–primer VPg revealed the critical interactions longer be maintained. For foot-and-mouth disease virus, the an- involved in the positioning and addition of the first nucleotide tiviral analogs ribavirin and 5-fluorouracil have been shown to be (UMP) to the primer molecule, providing insights into the mutagenic, contributing to virus extinction through lethal mu- mechanism of initiation of RNA genome replication in picor- tagenesis. Here, we report the x-ray structure of four elongation naviruses (14). complexes of foot-and-mouth disease virus polymerase 3D ob- Here, we report the crystallographic analysis of four catalytic tained in presence of natural substrates, ATP and UTP, or muta- complexes of FMDV 3D involving different nucleotides or genic nucleotides, ribavirin triphosphate and 5-fluorouridine mutagenic nucleotide analogs: 3D⅐GCAUGGGCCC⅐ATP, triphosphate with different RNAs as template–primer molecules.
    [Show full text]