The objective of this research is to provide new information about the function and catalytic mechanism of DNA-dependent RNA polymerase. To obtain such information, we propose to apply a variety of physical and chemical techniques, including absorption spectroscopy, circular dichroism (CD), fluorescence, stopped-flow kinetics, chemical and photochemical modification. These methods will be applied to RNA polymerase itself and to its complexes with DNA, substrates, substrate analogs and specific inhibitors. The aspects of RNA polymerase mechanism which we intend to explore are: (1) Binding of template to RNA polymerase; (2) The nature and location of catalytic groups in the enzyme; (3) The initiation and elongation process. 1. Binding of template. The nature of the conformational changes in the DNA and protein associated with formation of both closed and open promoter sites will be explored. Cloned restriction fragments of promoter-containing regions will be used. Fluorescence-labeled RNA polymerase will be used in stopped-flow kinetics studies of DNA binding to polymerase. The role of the Sigma subunit in DNA binding will be tested by determining whether Sigma might bind to single-stranded DNA. 2. Nature and location of catalytic groups. Chemical modification studies will be aimed at determining what groups participate in the catalysis. Nitration of tyrosine residues will be studied, especially those on the Sigma subunit. The site of affinity labels and photoaffinity labels will be determined in the sequence by chain separation cleavage and peptide mapping. Photooxidation with noncovalently and covalently attached dyes will be studied. 3. Initiation and elongation. The binding of NTPs and initiating dinucleotides to the enzyme and to enzyme-DNA complexes will be studied by spectroscopic methods. Specific promoter-containing fragments will be transcribed to a well-defined point to examine conformational differences between initiation and elongation complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023697-14
Application #
3271846
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1979-06-01
Project End
1993-06-30
Budget Start
1991-07-01
Budget End
1993-06-30
Support Year
14
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
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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