We have compiled and aligned the DNA sequences of 554 promoter regions from Escherichia coli and analysed the alignment for sequence similarities. We have focused on the similarities and differences between promoters that either do or do not contain an extended -10 element. The distribution of -10 and -35 hexamer element sequences, the range of spacer lengths between these elements and the frequencies of occurrence of different nucleotides, dinucleotides and trinucleotides were investigated. Extended -10 promoters, which contain a 5'-TG-3' element, tend to have longer spacer lengths than promoters that do not. They also tend to show fewer matches to the consensus -35 hexamer element and contain short runs of T residues in the spacer region. We have shown experimentally that the extended -10 5'-TG-3' motif contributes to promoter activity at seven different promoters. The importance of the motif at different promoters is dependent on the sequence of other promoter elements.

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the -10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the -10 and -35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions -15 and -14. Promoter activity is greatest when the 'non-template' strand carries T and G at positions -15 and -14, respectively. Promoter activity can be further enhanced by a second T and G at positions -17 and -16, respectively, immediately upstream of the first 'TG motif'. Our results show that the base sequence of the DNA segment upstream of the -10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters' -10 elements. We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters.

The Escherichia coli Rsd protein forms 1 : 1 complexes with sigma(70) protein, which is the major factor in determining promoter recognition by RNA polymerase. Here we describe measurements of the levels of Rsd, RNA polymerase, sigma(70) and the alternative sigma(38) factor. Rsd levels are sufficient to sequester a significant proportion of sigma(70) and immunoaffinity pull-down experiments show that this occurs in stationary phase but not in exponentially growing cells. Rsd expression is controlled by two promoters, P1 and P2. Experiments with lac fusions show that the P2 promoter is stronger than P1, that P2 is active in all phases of growth, and that this accounts for the high levels of Rsd.

The Escherichia coli Rsd protein forms complexes with the RNA polymerase 70 factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative 38 factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with 70 . Our experiments support a model in which the role of Rsd is primarily to sequester 70 , thereby increasing the levels of RNA polymerase containing the alternative 38 factor.The requirement of factors for the recognition of bacterial promoters by RNA polymerase (RNAP) is well known (9). Most bacteria contain a principal factor which assures the expression of most genes and minor factors which are needed for the expression of subsets of genes, often in response to specific stresses (10). Thus, in Escherichia coli K-12, 70 , encoded by rpoD, is the principal factor, while six alternative factors are present at lower levels. One of these alternatives, 38 , encoded by rpoS, accumulates when cell growth ceases and in response to certain stresses, and is regarded as the stationary-phase factor (11). Although the role of 38 is understood, it is not clear how it captures sufficient RNAP in order to ensure expression of the 38 regulon. This is because 38 has a weaker affinity for RNAP than 70 , and its level is always less than that of 70 (14, 22). Studies from several laboratories have identified different factors that might favor the formation of RNAP containing 38 , and its activity, in stationary phase (7,8,17). One such factor, discovered by Jishage and Ishihama (15), is the Rsd protein (regulator of sigma D) that was found to accumulate in stationary phase and to bind to free 70 . Jishage and Ishihama (16) showed that Rsd could increase expression from the 38 -dependent bolA promoter and reduce expression from certain 70 -dependent promoters and argued that by sequestering 70 , Rsd permits 38 , and possibly other alternative factors, to access RNAP. Recent studies showed that, as well as forming a 1:1 complex with 70 , Rsd can also interact with RNAP in the absence of , raising the possibility that Rsd might also affect gene expression in E. coli by direct interactions with RNAP (13, 27). Thus, in this work, we used transcriptomics to assess possible roles for Rsd and genetic analysis to investigate the mechanism of action of Rsd. We also describe the use of the bacterial adenylyl cyclase two-hybrid system (BACTH; reference 18) to investigate Rsd-70 and Rsd-Rsd interactions. MATERIALS AND METHODSBacterial strains. The starting E. coli K-12 strains used in this work were MG1655 (2), the ⌬lac strain MC4100 (24), and the cya strain BTH101 (19). The method of Datsenko and Wanner (5) was used to delete the rsd gene of MG1655 and insert a ka...

Previous work has shown that the base sequence of the DNA segment immediately upstream of the 3 310 hexamer at bacterial promoters (the extended 3 310 element) can make a signi¢cant 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 c c 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 c c 70 subunit can a¡ect the recognition of di¡erent extended 3 310 element sequences. ß

Genetics and biochemistry have been exploited to investigate transcription activation by the Escherichia coli CRP (cAMP receptor protein) factor at promoters with a DNA site for CRP near position -41 and the effects of a second upstream-bound CRP molecule. We show that the upstream-bound CRP contributes to transcription activation by improving the recruitment of RNA polymerase.