Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant contai ning double-amino-acid substitutions (I198A and I202A) on the hydrophobic f ace of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor bot h in vivo and in vitro. Our data revealed that the Ts sigma(A) factor posse ssed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resu lted in a very low core-binding activity of the Ts sigma(A) factor and a ma rkedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, sugg esting that extensive aggregation of the Ts sigma(A) is the main trigger fo r the temperature sensitivity of B, subtilis DB1005, Partial proteolysis, t ryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyse s revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exp osed on the protein surface, This hydrophobic exposure provides an importan t cue for mutual interaction between molecules of the Ts sigma(A) and allow s the formation of multimeric Ts sigma(A). Our results also indicate that I le-198 and Ile-202 on the hydrophobic face of the promoter -10 binding heli x are essential to ensure the correct folding and stabilization of the func tional structure of sigma(A) factor. (C) 2000 Wiley-Liss, Inc.