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

Area of Science:

• Microbiology
• Molecular Biology
• Protein Structure

Background:

• Sigma(A) factor is crucial for bacterial transcription initiation.
• Temperature-sensitive mutants provide insights into protein function and stability.

Purpose of the Study:

• To investigate the structural and functional basis of temperature sensitivity in Bacillus subtilis DB1005.
• To elucidate the role of specific amino acid substitutions in sigma(A) factor stability and activity.

Main Methods:

• In vivo and in vitro analyses of the mutant sigma(A) factor.
• Partial proteolysis, tryptophan fluorescence, and 1-anilinonaphthalene-8-sulfonate binding assays.
• Structural and functional characterization of Bacillus subtilis DB1005.

Main Results:

• The temperature-sensitive sigma(A) factor (Ts sigma(A)) forms multimeric structures prone to aggregation at restrictive temperatures.
• Extensive aggregation reduces core-binding activity and sigma(A)-RNA polymerase activity.
• Hydrophobic residues (Ile-198 and Ile-202) are essential for correct folding and stability.

Conclusions:

• Aggregation of the Ts sigma(A) factor is the primary cause of temperature sensitivity in Bacillus subtilis DB1005.
• Hydrophobic exposure on the promoter -10 binding helix drives multimerization and aggregation.
• Specific mutations disrupt the functional structure of the sigma(A) factor.