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|標題:||Differential and additive effects of the three conserved isoleucine residues in the promoter - 10 binding region on Bacillus subtilis sigma(A) structure and function||作者:||Liao, C.T.
|關鍵字:||alpha-helix;Bacillus subtilis;hydrophobic amino acids;sigma factor;suppressor mutant;polymerase chain-reaction;rna-polymerase;genetic-evidence;transcription initiation;overlap extension;ribonucleic-acid;sigma-70;subunit;groesl operon;a factor;recognition||Project:||Journal of Biochemistry||期刊/報告no：:||Journal of Biochemistry, Volume 126, Issue 3, Page(s) 461-469.||摘要:||
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.
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