Activation of the Bacillus subtilis spoIIG promoter requires interaction of Spo0A and the sigma subunit of RNA polymerase

Abstract

A's ability to direct transcription in vivo and in vitro. We found that alanine substitutions at these positions specifically reduced expression from the A -dependent, Spo0A-dependent promoters, spoIIG and spoIIE, in vivo. Furthermore, we found that stimulation of spoIIG promoter activity by Spo0A in vitro was reduced by the single substitutions H359A and H359R in A .A growing body of evidence supports the idea that sitespecific DNA binding proteins activate promoters in bacteria by contacting RNA polymerase (reviewed in reference 4). The site on RNA polymerase that is contacted by the activator protein appears to depend in part on the site on DNA at which the activator protein is bound (16). The Escherichia coli cyclic AMP receptor protein (CAP) interacts with the C-terminal domain (CTD) of the RNA polymerase alpha subunit when CAP is bound at a site centered about 61 bp upstream from the transcription start point (Ϫ61) (reviewed in reference 11), whereas CAP bound at a site centered at about position Ϫ41 activates transcription by contacting the N-terminal domain of the ␣ subunit (25; reviewed in reference 5). Other activator proteins contact the subunit of RNA polymerase. FNR (a protein that is structurally similar to CAP), PhoB, MalT, and cI from phage lambda bind to sites that overlap the Ϫ35 region of promoters, and probably contact the subunit of RNA polymerase (reference 5 and references therein; 9, 22-24). Some promoters contain multiple binding sites for activators. In some of these cases the two coactivators appear to interact directly with the RNA polymerase (6,18,29). At other promoters one coactivator affects DNA binding by the second coactivator, which is the only one to make direct contact with RNA polymerase (26).Spo0A from Bacillus subtilis, which is required for the initiation of sporulation, binds the spoIIG promoter at two sites known as 0A boxes, centered at positions Ϫ37 and Ϫ87 (reviewed in reference 15; 1, 27). Binding of Spo0A at these sites stimulates utilization of the spoIIG promoter by RNA polymerase containing A , a homolog of E. coli 70 (1, 3, 20, 27, 28). It is not known whether spoIIG promoter activation involves interactions between Spo0A and RNA polymerase. Baldus et al. (2) found that spoIIG promoter activity was reduced in mutants of B. subtilis in which A contained one of two single amino acid substitutions (glutamate substituted for lysine at position 356, K356E, or arginine substituted for histidine at position 359, H359R). These observations led to the suggestion that spoIIG promoter activation involves an interaction between Spo0A and A . The two amino acid substitutions in A that reduced spoIIG promoter activity lie near the region of A that makes sequence-specific contacts with the base pairs at the Ϫ35 region of its cognate promoters (Fig. 1). Other amino acid substitutions in this region of A and other sigma factors have been shown to change the specificity of promoter utilization by RNA polymerase containing these mutant sigma factors (12,19,32). An alternative e...

Phylogeny and functional conservation of σE in endospore-forming bacteria The GenBank accession numbers for the sequences determined in this work are AF225461–AF225466.

“…These results support previous reports of the ability of low levels of spoIIG activity (" 17 % of normal) to permit sufficient formation of endospores to allow spore detection under phase-contrast microscopy and production of viable colonies following chloroform treatment of 1-d-old cultures (Schyns et al, 1997). The absence of full recovery of the wild-type phenotype by the two complemented strains, and particularly by the spoIIG Bscomplemented strain, is likely a consequence of our extrachromosomal complementation strategy.…”

Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

“…2). However, since sporulation efficiency is not decreased significantly unless spoIIE (21) and spoIIG (9,40) transcription is reduced to less than 10% of that of the wild type, this result is consistent with the Spo ϩ phenotype of the strain.…”

Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis

“…We determined the nucleotide sequences of the spo0A and sigA regions for these six strains and found a single base pair substitution, a transition, that changed codon 355 (TGG) of the sigA gene, encoding arginine, to CGG, which encodes tryptophan. This allele was reconstructed in vitro by site-directed PCR mutagenesis, with either pJB2wtsigA, pJB2sigAH359R, or pJB2sigAH359A as template (1,4,25). The presence of the single or double mutations was confirmed by sequencing of the sigA allele in each plasmid with a Sequenase kit from Amersham.…”