Activating transcription in bacteria.

Bacteria use a variety of mechanisms to direct RNA polymerase to specific promoters in order to activate transcription in response to growth signals or environmental cues. Activation can be due to factors that interact at specific promoters, thereby increasing transcription directed by these promoters. We examine the range of architectures found at activator-dependent promoters and outline the mechanisms by which input from different factors is integrated. Alternatively, activation can be due to factors that interact with RNA polymerase and change its preferences for target promoters. We summarize the different mechanistic options for activation that are focused directly on RNA polymerase.

Analysis of mechanisms of activation and repression at bacterial promoters.

In bacteria, the expression of transcription units is controlled by regulatory regions, that contain one or more promoters and binding sites for regulatory proteins that activate or repress expression in response to different signals. In this chapter, we explain the diverse approaches that can be used to understand the mechanisms by which the different factors intervene, and how the effects are integrated. Bioinformatics, genetics and biochemistry must be combined to understand the organisation of regulatory regions and the mechanisms by which transcription initiation is controlled.

Identification of the CRP regulon using in vitro and in vivo transcriptional profiling.

The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons.

Identification and analysis of 'extended -10' promoters in Escherichia coli.

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.

Identification of functional regulatory regions of the connexin32 gene promoter.

Connexin32 (Cx32) is the predominant gap junction protein expressed in adult rat hepatocytes. This study investigated transcriptional regulation of the rat Cx32 gene in MH(1)C(1) rat hepatoma cells using transient expression assays in conjunction with promoter mutagenesis and 5' nested deletion analysis. Site-directed mutagenesis of the -736 and -187 hepatocyte nuclear factor-1 (HNF-1) sites, the -196 and -116 Sp1 sites, and the -729 and -329 Yin Yang 1 (YY1) sites all significantly reduced promoter activity. We have defined the contribution of each individual site to promoter activity in the intact cell. A novel upstream region of the Cx32 promoter (-1042 to -758) was cloned and shown to contain negative regulatory elements. The transcription factors HNF-1 and Sp1 have important functional roles in the transcriptional regulation of basal and cell-specific Cx32 expression. The multifunctional transcription factor YY1 is also implicated.

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.