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.     

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.     

Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase

We have isolated spontaneous temperature-resistant revertants of a temperature-sensitive mutation (rpoD800) in the sigma subunit of E. coli K12 RNA polymerase. These revertants still contained the rpoD800 allele. They were mucoid, and sensitive to ultraviolet light and the radiomimetic agent nitrofurantoin, which are characteristics of lon mutants. One revertant, Tr29, was mapped to the lon region of the chromosome. Lon- rpoD800 double mutants were constructed, and were phenotypically indistinguishable from the spontaneous temperature-resistant revertant. It is the degradation-deficient property of lon mutants that is responsible for the suppression of the temperature-sensitive phenotype. We show that the rpoD800 sigma polypeptide is a substrate for the ion proteolytic system, and that mutations in lon decrease the rate of mutant sigma degradation. The rate of synthesis of mutant sigma was also affected in lon- strains. The net effect of lon-mutations was to increase the concentration of mutant sigma. We conclude that the temperature-sensitive phenotype results from insufficient concentration, rather than altered function, of the mutant protein.     

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.     

Substitutions in the Escherichia coli RNA polymerase sigma70 factor that affect recognition of extended -10 elements at promoters

Previous work has shown that the base sequence of the DNA segment immediately upstream of the -10 hexamer at bacterial promoters (the extended -10 element) can make a significant contribution to promoter strength. Guided by recently published structural information, we used alanine scanning and suppression mutagenesis of Region 2.4 and Region 3.0 of the Escherichia coli RNA polymerase sigma(70) subunit to identify amino acid sidechains that play a role in recognition of this element. Our study shows that changes in these regions of the sigma(70) subunit can affect the recognition of different extended -10 element sequences.     

Mutational analysis of the histidine operon promoter of Salmonella typhimurium.

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.     

Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori rpoD gene in E. coli.

We constructed and analyzed hybrid Escherichia coli-Helicobacter pylori rpoD genes in an E. coli rpoD mutant. It turned out that a hybrid consisting of E. coli rpoD with subdomain 4.2 of H. pylori rpoD (for -35 recognition) was functional. On the other hand, hybrids consisting of E. coli rpoD with domain 2 and the adjacent sequence of H. pylori rpoD (for core enzyme binding and -10 recognition) were non-functional. Intriguingly, a hybrid rpoD containing H. pylori subdomain 4.2 conferred higher activity for the H. pylori PureA as determined by xylE expression of PureA-xylE fusions, although the activity of the hybrid rpoD for the tac promoter was comparable to that of E. coli rpoD. The tsp of ureA in E. coli with the hybrid rpoD and E. coli rpoD were 15 and 17bp upstream from that in H. pylori, respectively. The comparison of PureA sequences in both E. coli and H. pylori indicated the existence of a -10 consensus sequence but little conservation of -35 sequences. Instead, the PureA in both H. pylori and E. coli contained an identical heptamer, GTTAATA, in the extended -35 region.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.