Activity of mutant sigma F proteins truncated near the C terminus

Abstract

(F', the product of the spoILAC gene of Bacilus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors ofB. subtUis 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 sponrlate and for their ability to transcribe from a promoter (spoIHIG) that is normally read by RNA polymerase bound to cf (Eu").The results indicate that a mutant o' 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 cF: 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%o: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO terminus.When placed under starvation conditions, cells of Bacillus subtilis initiate a developmental process that leads, after several hours, to the formation of heat-resistant endospores. Early in development, an asymmetric septum divides the sporulating cell into two compartments, the forespore and the mother cell, in which gene expression occurs differentially (15). As sporulation proceeds, the cells manufacture a succession of sigma factors, each of which directs the RNA polymerase to transcribe certain sporulation-specific genes (6,19,31). Because these sigma factors are not needed for growth, they can be altered without affecting the viability of the cell; they are therefore ideally suited to studies of structure and function. Moreover, because much of the amino acid sequence (and therefore presumably the structure) is conserved between different sigma factors, such studies of sporulation-specific factors may also provide valuable information about the major sigma factors that direct transcription during vegetative growth of B. subtilis and other bacteria (10,12,14). In the known sigma factors, much of the C-terminal one-fifth of the sequence is strongly conserved. This part of the protein contains a region generally believed to constitute a helix-turn-helix motif, which interacts with the -35 region of the cognate promoters (2, 9, 29), followed by a short region rich in basic residues. After this basic region, conservation of the sequence abruptly breaks down, so that the C-terminal tails of the proteins have no apparent similarity (Fig. 1A).The experiments described in this paper are concerned with the C-terminal...