Nucleic acid polymerases use a general acid for nucleotidyl transfer (original) (raw)
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Proceedings of the National Academy of Sciences, 2007
The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.
Journal of Molecular Biology, 2009
The nucleotidyl transfer reaction catalyzed by DNA polymerases is the critical step governing the accurate transfer of genetic information during DNA replication, and its malfunctioning can cause mutations leading to human diseases, including cancer. Here, utilizing ab initio quantum mechanical/molecular mechanical calculations with free-energy perturbation, we carried out an extensive investigation of the nucleotidyl transfer reaction mechanism in the well-characterized high-fidelity replicative DNA polymerase from phage T7. Our defined mechanism entails an initial concerted deprotonation of a conserved crystal water molecule with protonation of the γ-phosphate of the deoxynucleotide triphosphate (dNTP) via a solvent water molecule, and then the proton on the primer 3′-terminus is transferred to the resulting hydroxide ion. Subsequently, the nucleophilic attack takes place, with the formation of a metastable pentacovalent phosphorane intermediate. Finally, the pyrophosphate leaves, facilitated by the relay of the proton on the γ-phosphate to the α-β bridging oxygen via solvent water. The computed activation free-energy barrier is consistent with kinetic data for the chemistry step with correct nucleotide incorporation in T7 DNA polymerase. This variant of the water-mediated and substrate-assisted mechanism has features tailored to the structure of the T7 DNA polymerase. However, a unifying theme in the water-mediated and substrate-assisted mechanism is the cycling through crystal and solvent water molecules of the proton originating from the primer 3′-terminus to the α-β bridging oxygen of the deoxynucleotide triphosphate; this neutralizes the evolving negative charge as pyrophosphate leaves and restores the polymerase to its pre-chemistry state. These unifying features are likely requisite elements for nucleotidyl transfer reactions.
Journal of Biological Chemistry
Reverse transcriptase isolated from avian myeloblastosis virus (AMV) and Rauscher murine leukemia virus (RLV) were examined for their ability to catalyze polymerization, ribonuclease H, pyrophosphate exchange, and pyrophosphorolysis reactions. A detailed characterization and a study of requirements for the expression of pyrophosphate exchange and pyrophosphorolysis reactions indicated that a variety of RNA and DNA template-primers supported these catalytic reactions. Furthermore, hydrogen bonding of template to primer was essential, although RNA:RNA templateprimers, e.g. poly(rA)*(rU)8 or 70 S RNA-tRNA complex, were not utilized for these reactions. A M V enzyme required M g + , and RLV enzyme Mn2+, as the preferred divalent metal ion for the expression of these activities. Response of various catalytic reactions to site-specific inhibitors revealed that polymerization and pyrophosphate exchange reactions were susceptible to reagents that affected either the substrate or the template binding site, intrinsic zinc, or sulfhydryl groups. RNase H and pyrophosphorolysis activities, on the other hand, exhibited susceptibility only to the template site-specific reagent. We, therefore, conclude that RNase H and pyrophosphorolysis reactions are catalyzed through the template binding site while polymerization and pyrophosphate exchange reactions require additional participation of the substrate binding site, as well as that of intrinsic zinc and the presence of reactive sulfhydryl groups.
Biochemistry, 1979
The diastereomers of adenosine 5'-0-( 1-thiotriphosphate) (ATP&) and adenosine 5'-0-(2-thiotriphosphate) (ATPPS) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the S , diastereomer is a better initiator than the R, isomer. The diasteromers of 3'-uridyl 5'-adenosyl 0,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the R, diastereomer is a better initiator than D N A-dependent RNA polymerase from Escherichia coli mediates the transcription of DNA to RNA by catalyzing the polymerization of ribonucleoside triphosphates in the presence of DNA template . The DNA-directed synthesis of RNA by RNA polymerase may be considered to involve two kinds of phosphodiester bond-forming steps Krakow et al., 1976): (1) an initiation step, wherein a purine ribonucleoside triphosphate and another ribonucleoside triphosphate are coupled to give a dinucleoside tetraphosphate, and (2) an elongation step, wherein a ribonucleoside triphosphate is added to the 3'-
Journal of the American Chemical Society, 2013
DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3′ group, thus activating O3′ as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wildtype enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3′ proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3′ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3′. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3′ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.
Biochimica et biophysica acta, 1978
1. The capacity of two-component ribonucleotidyl transferase to catalyze pyrophosphorolysis of polyribonucleotides is studied. 2. It is shown that nucleoside diphosphates (NDP), not being substrates for the enzyme, activate both the synthesis and pyrophosphorolysis of polynucleotides by the enzyme. The concentration of NDP is important for this effect: with an increase of NDP concentration the rate of synthesis increases and reaches a plateau at 10(-5) M NDP, while the rate of pyrophosphorolysis, attaining maximal values at 10(-5)--10(-3) M NDP, decreases with a further increase of NDP concentration. 3. The possible biological role of two-component ribonucleotidyl transferase is discussed.
FEBS Letters, 1993
S-Triphosphates of 1-(2',3'-epithio-2'.3'-dideoxy-B_D-lyxofuranosyl)thymine, l-(2',3'-eptthio-~,~-dideoxy-~-D-ribofuranosyl)th~lne and 2',3'lyxoanhydrothymidine have been shown to be termmatton substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase 1 from E. co11 and DNA polymerase j3 from rat hver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase E from human placenta. Km values of ItTTP. rtTTP and laTTP incorporation mto the DNA chain during catalysts by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than K,,, value for dTTP Furthermore, V,,, values for modified substrates are only 2-3 times lower than b',,,',,, for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides wtth a flattened furanosyl rmg for DNA polymerase active sites.
RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase
Virology, 1996
We have expressed the putative RNA-dependent RNA polymerase encoded by the potyvirus tobacco vein mottling virus (TVMV) in Escherichia coli as a glutathione S-transferase fusion protein. As prepared, the fusion protein possessed the poly(U) polymerase activity that is a hallmark of other picornavirus-encoded polymerases. In addition, this protein was able to utilize full-length TVMV RNA as a template for RNA synthesis. A fusion protein containing a mutation in the highly conserved GDD motif of the polymerase (GDD r ADD) possessed 7% of the activity of the wild type. Our results confirm that the presumed polymerase encoded by TVMV is in fact an RNA-dependent RNA polymerase and that the GDD motif so widely seen in viral polymerases has an important function in the TVMV protein.
2018
RNA viruses cause a number of acute and chronic diseases including the common cold, severe acute respiratory syndrome (SARS), and a more recent outbreak of Middle East respiratory syndrome (MERS). Vaccines developed against viruses have saved many lives. A traditional vaccine is a preparation of killed microorganisms, live attenuated organism, or living fully virulent organisms that is administered to produce or artificially increase immunity to a disease. However, safety concerns and efficacy are potential problems for the further development of new vaccines. One promising strategy to develop vaccines is by targeting the virally encoded RNA-dependent RNA polymerase (RdRp). The RdRp is conserved in most RNA viruses and these enzymes share a conserved structure and catalytical residues. It has been determined that RdRp error rate relates to viral attenuation. A too accurate RdRp loses adaptability to the host environment; RdRp with higher error rate are also detrimental because the p...
Nucleic Acids Research, 1995
For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O-methyl-NTPs, and the products contained up to 58% 2'-O-methyinucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATPaS, 2'-O-methyl-GTPaS and 2'-O-methyl-CTPaS, whereas they were found with dATP, 2'-O-methyl-ATPaS and 2'-O-methyl-UTPacS. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented.