Bacteriophage T7 RNA Polymerase and Its Active-site Mutants

Osumi-Davis, Patricia A.; Sreerama, Narasimha; Volkin, David B.; Middaugh, C. Russell; Woody, Robert W.; Woody, A. Young M.

doi:10.1006/jmbi.1994.1205

Cited by 41 publications

(27 citation statements)

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“…In particular our results agree with former studies showing that the probability of abortion rises sharply after incorporation of uridines (Ling et al, 1989;Martin et al, 1988;Osumi-Davis et al, 1994), and it is noteworthy that dT residues are absent from the nontemplate strand of natural T7 ITSs (Dunn & Studier, 1983). This observation has been tentatively attributed to the weakness of the rU-dA base-pair, leading to a particularly high k diss at these positions (Martin & Tinoco, 1980).…”

Section: Effects Of the Use Of A Non-cognate Its On In Vitro Abortivesupporting

confidence: 93%

“…Presumably, the nascent mRNA is loosely bound to the polymerase-DNA complex during transcription of the ITS: the slower the enzyme, the longer the time it takes to transcribe the ITS, and the higher the probability that mRNA dissociates during this time. Speed can be decreased by active site mutations (Bonner et al, 1992(Bonner et al, , 1994Osumi-Davis et al, 1994) or by using non-saturating concentrations of NTPs (Bonner et al, 1994). Another parameter that affects the extent of abortive transcription is the sequence of the ITS.…”

Section: Introductionmentioning

confidence: 98%

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The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo

Lopez

Guillerez²,

Sousa³

et al. 1997

Journal of Molecular Biology

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Section: Effects Of the Use Of A Non-cognate Its On In Vitro Abortivesupporting

confidence: 93%

Section: Introductionmentioning

confidence: 98%

The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo

Lopez

Guillerez²,

Sousa³

et al. 1997

Journal of Molecular Biology

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“…T7 RNA and DNA polymerases provide two other exceptions to the rule. Only two active-site carboxylates have been identified in T7 RNA polymerase (27,35). These two residues (Asp-537 and Asp-812) are far apart in the protein sequence, but both are important for metal binding and catalysis (36), and there is no candidate for a third carboxylate nearby in the crystal structure (37).…”

Section: Resultsmentioning

confidence: 99%

The CCA-adding Enzyme Has a Single Active Site

Yue¹,

Weiner²,

Maizels³

1998

Journal of Biological Chemistry

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The CCA-adding enzyme (tRNA nucleotidyltransferase) synthesizes and repairs the 3-terminal CCA sequence of tRNA. The eubacterial, eukaryotic, and archaeal CCA-adding enzymes all share a single active-site signature motif, which identifies these enzymes as belonging to the nucleotidyltransferase superfamily. Here we show that mutations at Asp-53 or Asp-55 of the Sulfolobus shibatae signature sequence abolish addition of both C and A, demonstrating that a single active site is responsible for addition of both nucleotides. Mutations at Asp-106 (and to a lesser extent, at Glu-173 and Asp-215) selectively impaired addition of A, but not C. We have previously demonstrated that the tRNA acceptor stem remains fixed on the surface of the CCA-adding enzyme during C and A addition (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Taken together with this new evidence that there is a single active site for catalysis, our data suggest that specificity of nucleotide addition is determined by a process of collaborative templating: as the single active site catalyzes addition of each nucleotide, the growing 3-end of the tRNA would progressively refold to create a binding pocket for addition of the next nucleotide.

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“…These data suggest that Arg-754 and Lys-758 in E. coli pol I are involved in catalysis but they do not establish a direct interaction with the dNTP or involvement in pyrophosphate release. More direct evidence stems from crystallographic data that showed that the Klenow fragment is complexed with pyrophosphate near the conserved Lys and Arg residues (42). Thus, our low fidelity mutants containing substitutions near Lys-663 or Arg-659 may exhibit attenuated pyrophosphate release.…”

Section: Low Fidelity Mutants Of T Aquaticus Dna Polymerase Imentioning

confidence: 96%

Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

Suzuki¹,

Avicola²,

Hood

et al. 1997

Journal of Biological Chemistry

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We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9670 -9675) by substituting an oligonucleotide containing random sequences for codons 659 -671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature-sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29 -68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZ␣ forward mutation assay, A661E and T664R yielded mutation frequencies at least 7-and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis. Thermus aquaticus (Taq) DNA polymerase I (pol I)1 is used extensively in the amplification of DNA by the polymerase chain reaction. Based on its primary sequence, crystal structure, and catalytic mechanism, Taq pol I is classified in the same family as Escherichia coli pol I (1-3). The amino acid sequences of Taq pol I and E. coli pol I are 38% homologous, both enzymes have 5Ј-3Ј exonuclease domains (4, 5), and the crystal structures of the polymerase domains are nearly identical (5, 6). The structure of the vestigial 3Ј-5Ј exonuclease domain of Taq pol I is very different, however; unlike E. coli pol I, purified Taq pol I exhibits no proofreading activity (7).The O-helix in E. coli pol I is constituted of 15 amino acids which form part of the DNA binding cleft (8). Crystallographic, biochemical, and mutagenesis studies indicate that four conserved residues in the O-helix which face toward the cleft interact with either the incoming dNTP or the templateprimer. Considerable evidence indicates that in E. coli pol I, Arg-754 and Lys-758 interact with the phosphate groups in the incoming dNTP, that Phe-762 interacts with the deoxyribose moiety in the dNTP, and that Tyr-766 binds to the templateprimer (9 -11). The nearly identical structures of the polymerase domains of Taq pol I and E. coli pol I (6, 7) suggest that the corresponding amino acids in Taq pol I have analogous functions. By using random sequence mutagenesis together with genetic complementation, we recently observed that the Ohelix of Taq pol I contains four amino acids which are immutable (or nearly immutable) and are apparently essential for catalysis in vivo (12). These residues, Arg-659, Lys-663, Phe-667, and Tyr-671, corres...

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Bacteriophage T7 RNA Polymerase and Its Active-site Mutants

Cited by 41 publications

References 0 publications

The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo

The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo

The CCA-adding Enzyme Has a Single Active Site

Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

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