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Suppression of TGA mutations in the Bacillus subtilis spoIIR gene by prfB mutations
M L Karow1, E J Rogers, P S Lovett
1Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.
Insights
Researchers discovered a high frequency of TGA nonsense mutations in Bacillus subtilis. Suppressor mutations in the prfB gene were identified, affecting gene expression and protein synthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Nonsense mutations, specifically TGA codons, can prematurely terminate protein synthesis.
- Identifying genetic suppressors is crucial for understanding gene function and regulatory mechanisms.
Purpose of the Study:
- To investigate the frequency and nature of chemically induced mutations in the Bacillus subtilis spoIIR locus.
- To identify and characterize suppressors of TGA nonsense mutations.
- To elucidate the role of the prfB gene in TGA mutation suppression and gene expression.
Main Methods:
- Chemical mutagenesis of Bacillus subtilis.
- Isolation and characterization of TGA nonsense mutations.
- Genetic mapping of suppressor mutations to the prfB gene.
- DNA sequencing of the prfB gene and revisions of its published sequence.
- Analysis of gene expression levels using reporter assays.
- N-terminal protein sequencing to identify inserted amino acids.
Main Results:
- A high proportion of TGA nonsense mutations were identified in the spoIIR locus.
- Six suppressors of TGA mutations were isolated, with five mapping to the prfB gene.
- This represents the first identification of mutations within the Bacillus subtilis prfB locus.
- The prfB mutations modulated the expression of a TGA-containing reporter gene (catA86-TGA) from 19% to 54%.
- N-terminal sequencing confirmed that tryptophan was inserted at the UGA codon due to prfB mutations.
Conclusions:
- The prfB gene plays a significant role in suppressing TGA nonsense mutations in Bacillus subtilis.
- prfB mutations can restore gene expression by facilitating readthrough of TGA codons.
- The findings provide new insights into translational termination and readthrough mechanisms in bacteria.
- Revisions to the prfB gene sequence were necessary, highlighting the importance of accurate genetic information.