Structural basis of DNA recognition by the alternative sigma-factor, sigma54

The sigma subunit of bacterial RNA polymerase (RNAP) regulates gene expression by directing RNAP to specific promoters. Unlike sigma(70)-type proteins, the alternative sigma factor, sigma(54), requires interaction with an ATPase to open DNA. We present the solution structure of the C-terminal domain of sigma(54) bound to the -24 promoter element, in which the conserved RpoN box motif inserts into the major groove of the DNA. This structure elucidates the basis for sequence specific recognition of the -24 element, orients sigma(54) on the promoter, and suggests how the C-terminal domain of sigma(54) interacts with RNAP.     

Mutational analysis of an extracytoplasmic-function sigma factor to investigate its interactions with RNA polymerase and DNA

The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.     

The SigB sigma factor mediates high-temperature responses in the cyanobacterium Synechocystis sp. PCC6803

The sigma factors of RNA polymerase play central roles when bacteria adapt to different environmental conditions. We studied heat-shock responses in the cyanobacterium Synechocystis sp. PCC6803 using the sigma factor inactivation strains deltasigB, deltasigD and deltasigBD. The SigB factor was found to be important for short-term heat-shock responses and acquired thermotolerance. The normal high-temperature induction of the hspA gene depended on the SigB factor. The SigD sigma factor had a role in high-temperature responses as well, and the double inactivation strain deltasigBD grew more slowly at 43 degrees C than the deltasigB and deltasigD strains.     

Two functional domains conserved in major and alternate bacterial sigma factors

Sequences of the sigma factors of Escherichia coli and Bacillus subtilis were aligned with the sequences of two sigma-like proteins, HtpR, involved in the expression of heat-shock genes in E. coli, and SpoIIG, necessary for endospore formation in B. subtilis. An internal region is highly conserved in the four proteins and is proposed to be involved in binding of sigma factors to core RNA polymerase. The carboxy-terminal part of the four proteins presents the characteristic structure found in several prokaryotic DNA-binding proteins and is proposed to be involved in promoter recognition.     

The molecular basis of selective promoter activation by the sigmaS subunit of RNA polymerase

Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, sigmaS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, sigma70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing sigmaS or sigma70, or by both holoenzymes. How these promoter features impose sigmaS selectivity is further discussed. Moreover, additional pathways allow sigmaS to compete more efficiently than sigma70 for limiting amounts of core RNAP (E) and thereby enhance EsigmaS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes.     

Inhibitory effect of phosphate on in vitro development of 2-cell rat embryos is overcome by a factor(s) in oviductal extracts

The promoter recognition site on the sigma70 initiation factor is shielded from interaction with DNA unless sigma70 is bound to the core component of RNA polymerase (RNAP). It is shown that interaction of sigma70 with the isolated beta' subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site. Using UV-induced DNA-protein cross-linking we demonstrate that free beta' stimulates specific cross-links between region 2 of the sigma70 polypeptide and a fragment of the non-template promoter strand containing the TATAAT sequence. Thus the sigmabeta' subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core.