The major goal of the work proposed in this application is the investigation of the structure-function relationships of the Beta and Beta' subunits of core RNA polymerase of E. coli by a genetic, physiological and biochemical analysis of mutants. Five different classes of RNA polymerase mutants will be chosen for intensive analysis: rifampicin resistant mutants, streptolydigin resitant mutants, mutants with altered sigma interaction, mutants with altered DNA binding and mutants that exhibit a dominant lethal phenotype. We will use deletion mapping to locate precisely the mutations within rpoB (coding for Beta) or rpoC (coding for Beta'). In vivo analysis of regulation of gene activity and in vitro analysis of DNA binding and transcription will enable us to assess the nature of the functional alteration in the mutant RNA polymerase. Based on such studies, we will be able to determine whether mutations resulting in the same or similar functional alterations are clustered on the genetic map. DNA sequencing of clustered mutations will enable us to correlate functional defect with changes in primary and secondary structure of the protein. Finally, second site revertants of some mutant classes will be used to identify factors which interact with core polymerase.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019635-05
Application #
3128974
Study Section
(MG)
Project Start
1983-01-01
Project End
1987-11-30
Budget Start
1987-01-01
Budget End
1987-11-30
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Dombroski, A J; Johnson, B D; Lonetto, M et al. (1996) The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing. Proc Natl Acad Sci U S A 93:8858-62
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Heisler, L M; Suzuki, H; Landick, R et al. (1993) Four contiguous amino acids define the target for streptolydigin resistance in the beta subunit of Escherichia coli RNA polymerase. J Biol Chem 268:25369-75
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Dombroski, A J; Walter, W A; Record Jr, M T et al. (1992) Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Cell 70:501-12
Jin, D J; Gross, C A (1991) RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation. J Biol Chem 266:14478-85
Hager, D A; Jin, D J; Burgess, R R (1990) Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase. Biochemistry 29:7890-4

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