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Structural and functional properties of a Bacillus subtilis temperature‐sensitive σ A factor
Author(s) -
Wen YuDer,
Liao ChaoTsai,
Liou KungMing,
Wang WenHorng,
Huang WeiCheng,
Chang BanYang
Publication year - 2000
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/1097-0134(20000901)40:4<613::aid-prot60>3.0.co;2-k
Subject(s) - chemistry , proteolysis , bacillus subtilis , biophysics , mutant , folding (dsp implementation) , biochemistry , helix (gastropod) , hydrophobic effect , tryptophan , amino acid , biology , enzyme , genetics , gene , ecology , snail , bacteria , electrical engineering , engineering
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 σ A factor. We have analyzed the structural and functional properties of this mutant σ A factor both in vivo and in vitro. Our data revealed that the Ts σ A factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts σ A resulted in a very low core‐binding activity of the Ts σ A factor and a markedly reduced σ A ‐RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts σ A 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 σ A factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts σ A and allows the formation of multimeric Ts σ A . 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 σ A factor. Proteins 2000;40:613–622. © 2000 Wiley‐Liss, Inc.