sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA.
Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.
Huang
X
X
Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.
Lopez de Saro
F J
FJ
Helmann
J D
JD
D001412:Bacillus subtilis / Q000201:enzymology; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D019077:Helix-Turn-Helix Motifs; D009154:Mutation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D012808:Sigma Factor; D017868:Cyclic AMP-Dependent Protein Kinases; D012321:DNA-Directed RNA Polymerases