The importance of region 2.1 in sustaining the functional structure of the Bacillus subtilis sigma(A) factor.
Region 2.1 of the sigma factor is once proposed to be involved in core binding, and certain bulky hydrophobic amino acids in region 2.1 are thought to make contact with the conserved isoleucine residues in the promoter -10 binding region on the same protein. To examine the roles of the contact between these two regions in sigma(A) structure and function, sigma(A )factor with L145A, I149A, or Y153A was created, and the effects of each substitution on the growth of Bacillus subtilis and on the structural and functional properties of sigma(A) were analyzed. Our data revealed that the growth potential of B. subtilis was significantly affected by each of the substitutions of sigma(A) at elevated temperature. The growth defect was most pronounced with the strain containing L145A-sigma(A); it possessed a low growth potential even at 37 degrees C. In parallel, changes in the structural stability and core-binding activity of sigma(A) and in the promoter-binding and transcription activities of sigma(A)-RNA polymerase were observed for each of the substitutions, with the most drastic effects exerted by L145A. Clearly, region 2.1 of sigma(A) has extra functions, such as the binding of RNA polymerase to promoter DNA, other than the known core-binding ability. Moreover, the multiple effects of each of the substitutions on sigma(A) demonstrate that the contacts between the hydrophobic amino acids in region 2.1 and those in the promoter -10 binding region are critical to the maintenance of the functional sigma(A) structure and that L145 in region 2.1 plays an important role in this respect.
Liao
Chao-Tsai
CT
Chungtai Institute of Health Sciences and Technology, Taichung 40227, Taiwan, Republic of China.
Liao
Hsuen-Chun
HC
Huang
Wei-Cheng
WC
Wang
Wen-Horng
WH
Chang
Ban-Yang
BY
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004586:Electrophoresis / Q000379:methods; D006207:Half-Life; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013329:Structure-Activity Relationship; D013696:Temperature; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012808:Sigma Factor; D007532:Isoleucine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases