Evidence that the transcriptional activator Spo0A interacts with two sigma factors in Bacillus subtilis

Abstract

The transcriptional regulator Spo0A activates transcription from two types of promoters. One type of promoter is used by RNA polymerase containing sigma A, whereas the other type is used by RNA polymerase containing sigma H. There are Spo0A-binding sites near the -35 region of both types of promoters. It has been reported that some transcriptional regulators that bind near the -35 regions of promoters directly interact with the sigma subunit of RNA polymerase. Therefore, we looked for evidence that Spo0A interacts with both sigma factors by searching for single amino acid substitutions in these factors that specifically prevent expression from Spo0A-dependent promoters, but that do not decrease activity of Spo0A-independent promoters. Two such amino acid substitutions were isolated in sigma A and one was isolated in sigma H. The amino acid substitutions in sigma A prevented expression from the Spo0A-activated promoters, spoIIG and spoIIE, but expression was not impaired from the Spo0A-independent, sigma A-dependent promoter tms or from the Spo0A-activated, sigma H-dependent promoter, spoIIA. The amino acid substitution in sigma H prevented expression from the spoIIA promoter but not from the Spo0A-independent promoter, citGp2, which is used by sigma H-RNA polymerase. All of these amino acid substitutions occur in the carboxyl terminus of the sigma factors. These amino acid substitutions may define the sites of contact between the sigma factors and Spo0A. The ability of response regulators such as Spo0A to interact with multiple sigma factors may increase the variety of responses made by bacteria using a limited number of transcription factors.

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

“…However, these substitutions did not affect the utilization of A -dependent, Spo0A-independent promoters or H -dependent, Spo0A-dependent promoters. Alanine substitutions at these sites in A produced similar effects (1). Therefore, these substitutions define positions in region 4 of A that are essential specifically for Spo0A-dependent activation of the spoIIG and spoIIE promoters.…”

“…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.…”

“…Spo0A may activate the A -dependent promoters by interacting directly with the A subunit of the RNA polymerase. Baldus et al (1) found that spoIIG and spoIIE promoter activity was reduced in mutants of B. subtilis in which A contained one of two single amino acid substitutions in region 4, with the lysine at position 356 replaced by glutamate (K356E) or the histidine at 359 replaced by arginine (H359R). However, these substitutions did not affect the utilization of A -dependent, Spo0A-independent promoters or H -dependent, Spo0A-dependent promoters.…”

SpoOA represses transcription of the cry toxin genes in Bacillus thuringiensis

“…The OH-associated RNA polymerase transcribes genes during the transition phase in B. subtilis; the regulatory protein SpoOA interacts with both oA-and oH-dependent promoters, acting as either a negative or a positive effector of gene expression (Baldus et al, 1994(Baldus et al, , 1995.…”

Transcription activation by phage phi29 protein p4 is mediated by interaction with the alpha subunit of Bacillus subtilis RNA polymerase.

“…The sigma factor determines the promoter specificity of RNAP and also interacts with certain transcriptional activators (9)(10)(11)(12)(13)(14). The elongation of the transcript is undertaken by the core enzyme (a21313').…”