Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) 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 sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(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 sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(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 sigma(A) factor.
Wen
Y D
YD
Institute of Biochemistry, National Chung-Hsing University, Taiwan, Republic of China.
Liao
C T
CT
Liou
K M
KM
Wang
W H
WH
Huang
W C
WC
Chang
B Y
BY
D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014158:Transcription, Genetic
D012808:Sigma Factor