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Mutation changing the specificity of an RNA polymerase sigma factor
1Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138.
Journal of Molecular Biology
|April 20, 1989
Summary
A Bacillus subtilis mutation alters the sigma H RNA polymerase, decreasing its ability to transcribe wild-type promoters while enhancing transcription of mutant promoters. This discovery sheds light on sigma factor-promoter interactions.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacillus subtilis utilizes alternative RNA polymerase holoenzymes for gene regulation during sporulation.
- Sigma H is a key sigma factor directing transcription of the sporulation gene spoVG.
Purpose of the Study:
- To characterize a mutation in the spo0H gene affecting sigma H RNA polymerase specificity.
- To understand the molecular basis of promoter recognition by sigma factors.
Main Methods:
- Site-directed mutagenesis of the spo0H gene.
- In vitro transcription assays using wild-type and mutant RNA polymerase holoenzymes.
- Analysis of transcription from wild-type and mutant spoVG promoters.
Main Results:
- The spo0H81 mutation specifically altered sigma H RNA polymerase, blocking wild-type spoVG promoter transcription.
- This mutation enhanced transcription from a mutant spoVG promoter (spoVG249) with a -10 region mutation.
- The spo0H81 mutation resulted in a threonine to isoleucine substitution at position 100 in sigma H.
Conclusions:
- The spo0H81 mutation confers a change-of-specificity, altering sigma H's promoter recognition capabilities.
- The conserved region in sigma H, particularly Thr100, may be involved in direct contact with the promoter's -10 region.