Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition

We describe a mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase, which alters the promoter specificity of the holoenzyme in vivo. The mutant sigma causes a substantial and specific increase in the activity of mutant ant and lac promoters with a T.A to C.G substitution at position -12, the first position of the -10 hexamer. The rpoD mutation is a single base-pair substitution causing a Gln----His change at position 437, which is in a domain of conserved region 2.4 that is predicted to form an alpha-helix. Gln437 would lie one turn of the alpha-helix away from Thr440, which was previously implicated in recognition of position -12. The rpoD-QH437 mutation described here lends further support to the model that region 2.4 of sigma is involved in recognition of the 5' end of the -10 hexamer. In addition, two rpoD mutations with non-specific effects on promoter recognition are described.     

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.     

Principal sigma subunit of the Caulobacter crescentus RNA polymerase.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.     

On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter

Bacterial promoters of the extended -10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended -10 Escherichia coli galP1 promoter at temperatures as low as 6 degrees C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended -10 motif and/or containing the -35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif. However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent. Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif. We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations.     

Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection

The DNase I protection pattern of E sigma 32 was assayed on three heat shock promoters, the E sigma 32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the E sigma 32 promoter upstream from rpoD. E sigma 32 protected each of these promoters from DNase I digestion from around -60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. E sigma 32 binding altered the sensitivity to methylation of bases in the vicinity of both the -10 and -35 regions. The DNase I footprints for the E sigma 32 promoters were very similar to the DNase I footprint of E sigma 70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that E sigma 32, like E sigma 70, contacts the DNA primarily on one face of the helix in the -35 region and on both faces in the -10 region.     

Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase.

Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.