Structural and functional properties of a Bacillus subtilis temperature-sensitive σA factor

Bacillus subtilis DB1005 is a temperature‐sensitive (Ts) sigA mutant containing double‐amino‐acid substitutions (I198A and I202A) on the hydrophobic face of the promoter −10 binding helix of σA factor. We have analyzed the structural and functional properties of this mutant σA factor both in vivo and in vitro. Our data revealed that the Ts σA factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts σA resulted in a very low core‐binding activity of the Ts σA factor and a markedly reduced σA‐RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts σA is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1‐anilinonaphthalene‐8‐sulfonate–binding analyses revealed that the hydrophobic face of the promoter −10 binding helix and also the hydrophobic core region of the Ts σA factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts σA and allows the formation of multimeric Ts σA. Our results also indicate that Ile‐198 and Ile‐202 on the hydrophobic face of the promoter −10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of σA factor. Proteins 2000;40:613–622. © 2000 Wiley‐Liss, Inc.