The interaction of Bacillus subtilis sigmaA with RNA polymerase.
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 sigma factors, making the essential interaction between sigma 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 sigma factor can be disrupted by introducing carefully designed mutations into sigma(A) of Bacillus subtilis. This disruption was demonstrated qualitatively by Far-Western blotting. Design of mutant sigmas was achieved by computer-aided visualization of the RNAP-sigma interface of the B. subtilis holoenzyme (RNAP + sigma) 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-sigma interface model to be constructed for the design of mutations, a crucial error in the B. subtilis sigma(A) sequence in published databases at amino acid 165 had to be corrected first. The new model was validated through determination of RNAP-sigma interactions using targeted mutations.
Johnston
Elecia B
EB
Discipline of Biological Sciences, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Lewis
Peter J
PJ
Griffith
Renate
R
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015203:Reproducibility of Results; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D020035:Holoenzymes; D010455:Peptides; D012808:Sigma Factor; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases