δDifferential and Additive Effects of the Three Conserved Isoleucine Residues in the Promoter -10 Binding Region on Bacillus subtilis δ_A Structure and Function

Abstract

The promoter -10 binding region of the Bacillus subtilis δ^A factor forms an amphiphilic α-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 α-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 δ^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 δ^A; they affect differentially and additively the structure and function of δ^A, with the central isoleucine residue (Ile-198) playing the most important role. By analogy with the crystal structure of a δ⁷⁰ peptide, it is apparent that interdigital interactions exist between the three conserved isoleucine residues and certain hydrophobic amino acids in region 2.1 of δ^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 δ^A by repacking the hydrophobic core of δ^A. The second-site suppressor (S291F) appears to be allele-specific, but it is not as effective as the same-site suppressors in restoring δ^A structure and function.