The importance of a proper helical structure in the promoter-10 binding region to Bacillus subtilis sigma A structure and function.
Two Bacillus subtilis sigA mutants with amino acid substitutions tending to disrupt the structure of the promoter -10 binding helix of B. subtilis sigma A factor were constructed. B. subtilis DB1001 which contained an A197P substitution was very sensitive to temperature elevation. B. subtilis DB1002 had a T199G substitution and was low in growth potential at the elevated temperature. Degradation of sigma A in B. subtilis DB1001 (t(1/2)=63.2 min) and DB1002 (t(1/2)=186.0 min) occurred readily even at 37 degrees C; however, sigma A in B. subtilis DB2 (wild-type) was fairly stable at the same temperature. The activities of both DB1001 and DB1002 sigma A factors on groE promoter (sigma A-type) were lower than those of the wild-type counterpart at both permissive and restrictive temperatures. The failure of a higher sigma A concentration to suppress the Ts phenotype of DB1001 indicated that the temperature sensitivity of B. subtilis DB1001 was due to altered function, rather than insufficient concentration, of sigma A in the cells. Taken together, our results suggest that the helicity of the promoter -10 binding helix is essential to the packing interaction in the hydrophobic core region of sigma A, which helps to maintain the stable and functional sigma A structure.
Liao
C T
CT
Agricultural Biotechnology Laboratories, National Chung-Hsing University, Taiwan.
Wen
Y D
YD
Wang
W H
WH
Tsai
S C
SC
Doi
R H
RH
Chang
B Y
BY
D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics; D006358:Hot Temperature; D016296:Mutagenesis; D010957:Plasmids / Q000138:chemical synthesis; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D018833:Chaperonins