Analysis of the distribution of the nucleotide sequence and electrostatic potential of the Escherichia coli genome

The oligonucleotide composition of the E. coli genome and its sigma70-specific promoters has been analyzed. The promoter DNA was shown to contain mainly AT-rich hexanucleotides having functionally important physical properties such as the ability to form easily melting sites and induce the bending of the double helix. A comparative analysis of the electrostatic characteristics of hexanucleotides within the whole sequence of the E. coli genome and its promoter regions was made. Hexanucleotides possessing a more electronegative surrounding were found to predominate in the nucleotide sequence of the promoter DNA.     

Role of the spacer between the -35 and -10 regions in sigmas promoter selectivity in Escherichia coli

In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.     

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.     

Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters

Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.     

Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella

Flagellar class 3 operons of Escherichia coli and Salmonella are transcribed by RNA polymerase containing sigma 28. The consensus sequence of the sigma 28-dependent promoters was believed to be TAAA N15 GCCGATAA. In this study, we found that the E. coli genome contains a large number of sequences homologous to this consensus. However, we showed that they do not always exert a sigma 28-dependent promoter activity. We compare more carefully the sequences of the class 3 flagellar promoters and propose a revised structure of the sigma 28-dependent promoters as TAAAGTTT N11 GCCGATAA.     

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.     

Sigma 28 promoter prediction in members of the Gammaproteobacteria

A two-step approach was used to predict sigma(28) promoter sequences in a number of Gammaproteobacteria. Putative promoters were initially identified upstream of motility and chemotaxis genes in test species based on their similarity to the Escherichia coli sigma(28) promoter consensus. These sequences were then used to generate position specific score matrices that were used iteratively during whole-genome analyses. The consistent identification of promoter sequences upstream of orthologous genes suggested that the predictions were biologically relevant.     

Cloning and characterization of the promoters of temperate mycobacteriophage L1

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the beta-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli sigma70-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early Pleft promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the Pleft equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli sigma70 promoters.