Differential and additive effects of the three conserved isoleucine residues in the promoter -10 binding region on Bacillus subtilis sigma(A) structure and function.
The promoter -10 binding region of the Bacillus subtilis sigma(A) factor forms an amphiphilic alpha-helix with three conserved isoleucines located at four-residue intervals. To investigate the structural and functional roles of the three isoleucine residues, we constructed a series of sigA mutants with single and double Ile-to-Ala substitutions on the hydrophobic face of this alpha-helix and isolated intragenic revertants with either same-site or second-site suppressor that partially restores the structural stability and transcription activity of the mutant sigma(A) factors. Our data show that the three conserved isoleucine residues (Ile-194, Ile-198, and Ile-202) are involved in the hydrophobic core packing of sigma(A); they affect differentially and additively the structure and function of sigma(A), with the central isoleucine residue (Ile-198) playing the most important role. By analogy with the crystal structure of a sigma(70) peptide, it is apparent that interdigital interactions exist between the three conserved isoleucine residues and certain hydrophobic amino acids in region 2. 1 of sigma(A). They include at least the van der Waals contacts between Ile-194 and both Leu-145 and Ile-149, between Ile-198 and both Ile-149 and Tyr-153, as well as between Ile-202 and Tyr-153. The same-site suppressors, Val-194 and Val-198, restore the structural stability and transcription activity of sigma(A) by repacking the hydrophobic core of sigma(A). The second-site suppressor (S291F) appears to be allele-specific, but it is not as effective as the same-site suppressors in restoring sigma(A) structure and function.
Liao
C T
CT
Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan.
Wang
W H
WH
Yang
H S
HS
Chen
J P
JP
Chang
B Y
BY
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012808:Sigma Factor; D007532:Isoleucine; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases