Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis.

The specificity of promoter binding by RNA polymerase is governed by the sigma subunit. Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters. We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis. These mutations change the specificity of sigma A, the primary sigma factor in growing B. subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70. These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo.     

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

The recognition and prediction of sigma70 promoters in Escherichia coli K-12

Based on the conservation analysis of the 683 latest experimentally verified sigma(70)-promoter sequences of Escherichia coli K-12, it is found that the conservative hexamers segments in different sites play a key role of promoter regions, a novel position-correlation scoring matrix (PCSM) algorithm for predicting sigma(70) promoter is presented. The predictive capacity of the algorithm is tested by 10-cross validation test. The results show that the overall prediction accuracies (sensitivity) and specificity are 91% and 81%, respectively. By selecting the 683 experimentally verified sigma(70) promoters as training set and searching for the complete sequence in E. coli K-12 with 4639221bp. Results show that the 100% of the 683 experimentally verified sigma(70) promoters have been identified and some possible promoters are predicted.     

Substitution of RNA-polymerase sigma-subunits and transcription regulation

Recent data on regulation of gene activity in bacteria by substitution of RNA polymerase sigma subunits are reviewed. The htpR gene which controls the switch-on of the Escherichia coli heat-shock protein synthesis codes for sigma 32 subunit. sigma 32-containing RNA polymerase transcribes the heat-shock genes in vitro from specific promoters of no use for RNA polymerase containing the major sigma 70 subunit. Several minor sigma subunits have been found in Bacillus subtilis vegetative cells, in addition to the major sigma 55 subunit, differing in the specificity of promoter recognition. Many B. subtilis genes are controlled by tandemly located promoters recognized by RNA polymerases carrying different sigma subunits. sigma 29 subunit is encoded by spoIIG gene and is probably involved in the regulation of sporulation. Specific sigma subunits for transcribing "middle" or "late" genes are encoded by a number of phages.     

Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma 32.

Escherichia coli K-12 strain 285c contains a short deletion mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase. The sigma 70 protein encoded by this allele (rpoD285) unstable, and this instability leads to temperature-sensitive growth. Pseudorevertants of 285c that can grow at high temperature contain mutations in the rpoH gene (encoding the heat shock sigma factor sigma 32), and their mutant sigma 70 proteins have increased stability. We characterized the alterations in three of these rpoH alleles. rpoH111 was a point mutation resulting in a single amino acid substitution. rpoH107 and rpoH113, which are known to be incompatible with rpoD+, altered the restriction map of rpoH. rpoH113 was deleted for 72 base pairs of the rpoH gene yet retained some sigma 32 activity. rpoH107 had two IS1 elements that flanked an unknown DNA segment of more than 6.4 kilobases inserted in the rpoH promoter region. The insertion decreased the amount of rpoH mRNA to less than 0.5% of the wild-type level at 30 degrees C. However, the mRNA from several heat shock promoters was decreased only twofold, suggesting that the strain has a significant amount of sigma 32.