Activity of mutant sigma F proteins truncated near the C terminus.
sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B. subtilis and Escherichia coli. However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable. It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor. We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus. We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F). The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable. By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein. When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.
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Department of Biochemistry, University of Oxford, United Kingdom.
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D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012808:Sigma Factor; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C064971:FliA protein, Bacteria; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012808:Sigma Factor; D001616:beta-Galactosidase