The overall objective of this research is to elucidate the catalytic mechanism of DNA-dependent RNA polymerase. In order to obtain such information, we propose to continue and extend our studies of T7 RNA polymerase in which functionally significant residues have been altered. By site-directed mutagenesis, we have mutated five sites which are strongly conserved among RNA and DNA polymerases and shown that D537 and D812 are essential for activity, and K631, Y639 and H811 are functionally significant. The role in the polymerase mechanism of each of these functional groups, and others to be identified by further mutagenesis, will be determined. Each step in the addition of single nucleotides to initiation and elongation complexes will be analyzed by pre-steady-state experiments utilizing abortive initiation, quench-flow, pyrophosphorolysis and pyrophosphate exchange, and fluorescence-detected stopped flow. Thio analogs of the nucleotides will be used to help distinguish between chemical and non-chemical (conformational changes and product dissociation) steps. A detailed comparison between the kinetic and equilibrium parameters for the wild-type and mutant enzymes will permit assignment of specific roles to each mutated side-chain. Specific experiments will test mechanistic hypotheses, e.g. the possibility that the two critical Asp residues bind divalent cations in a two-metal-ion mechanism as in DNA polymerase. Binding of Mn2+ to wild-type and mutants with replacements at D537 and D812 will be measured by EPR and flow dialysis. Mn2+in the presence of HCO3 will be used to catalyze the H202 oxidation of side chains near the metal binding site. These residues can be labeled by reduction with NaB3H4 and identified after digestion and peptide separation. Co2+ nucleotides will be crosslinked to wild-type and mutants by H202 oxidation. Further sites critical for RNA polymerase activity will be identified by cassette mutagenesis. It is these presently unidentified sites which are most likely to be unique to RNA polymerase as opposed to DNA polymerase. CD and fluorescence will be used to compare the conformation and stability of the mutants with the wild-type enzyme. Photoaffinity labeling with nascent RNAs in which 8-N3ATP has been incorporated will be used to identify regions of the enzyme with which the product RNA interacts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023697-16
Application #
2174147
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-06-01
Project End
1997-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
16
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Griko, Y; Sreerama, N; Osumi-Davis, P et al. (2001) Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study. Protein Sci 10:845-53
Woody, A Y; Osumi-Davis, P A; Hiremath, M M et al. (1998) Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F. Biochemistry 37:15958-64
Woody, A Y; Eaton, S S; Osumi-Davis, P A et al. (1996) Asp537 and Asp812 in bacteriophage T7 RNA polymerase as metal ion-binding sites studied by EPR, flow-dialysis, and transcription. Biochemistry 35:144-52
Lopez de Saro, F J; Woody, A Y; Helmann, J D (1995) Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase. J Mol Biol 252:189-202
Osumi-Davis, P A; Sreerama, N; Volkin, D B et al. (1994) Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization. J Mol Biol 237:5-19
Osumi-Davis, P A; de Aguilera, M C; Woody, R W et al. (1992) Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity. J Mol Biol 226:37-45
Knoll, D A; Woody, R W; Woody, A Y (1992) Mapping of the active site of T7 RNA polymerase with 8-azidoATP. Biochim Biophys Acta 1121:252-60
Shimer Jr, G H; Woody, A Y; Woody, R W (1988) Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects. Biochim Biophys Acta 950:354-65
Woody, A Y; Evans, R K; Woody, R W (1988) Characterization of a photoaffinity analog of UTP, 5-azido-UTP for analysis of the substrate binding site on E. coli RNA polymerase. Biochem Biophys Res Commun 150:917-24
Wheeler, A R; Woody, A Y; Woody, R W (1987) Salt-dependent binding of Escherichia coli RNA polymerase to DNA and specific transcription by the core enzyme and holoenzyme. Biochemistry 26:3322-30

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