The interaction of Bacillus subtilis sigmaA with RNA polymerase

Johnston, Elecia B.;Lewis, Peter J.;Griffith, Renate

Abstract

RNA polymerase (RNAP) is an essential and highly conserved enzyme in all organisms. The process of transcription initiation is fundamentally different between prokaryotes and eukaryotes. In prokaryotes, initiation is regulated by σ factors, making the essential interaction between σ factors and RNAP an attractive target for antimicrobial agents. Our objective was to achieve the first step in the process of developing novel antimicrobial agents, namely to prove experimentally that the interaction between a bacterial RNAP and an essential σ factor can be disrupted by introducing carefully designed mutations into σA of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant σs was achieved by computer-aided visualization of the RNAP-σ interface of the B. subtilis holoenzyme (RNAP + σ) constructed using a homology modeling approach with published crystal structures of bacterial RNAPs. Models of the holoenzyme and the core RNAP were rigorously built, evaluated, and validated. To allow a high-quality RNAP-σ interface model to be constructed for the design of mutations, a crucial error in the B. subtilis σA sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-σ interactions using targeted mutations.

Journal

N/A

Publication Name

N/A

Volume

ISBN/ISSN

1469-896X

Edition

N/A

Issue

Pages Count

Location

N/A

Publisher

Wiley

Publisher Url

N/A

Publisher Location

N/A

Publish Date

N/A

Url

N/A

Date

N/A

EISSN

N/A

DOI

10.1002/pro.239