Tight regulation of gene expression is crucial for the survival of an organism, and allows certain genes to be switched on or off depending on the growth conditions and the state of differentiation. Transcription initiation is the most highly regulated step of gene expression, preventing wastage and being subject to the action of sophisticated signalling pathways. The RNA polymerases are further regulated through multiple different activators (stimulating transcription) and repressors (inhibiting transcription). Notably, promoter recognition specificity in bacteria is regulated by several dissociable sigma factors which can bind to the RNA polymerase core enzyme. Sigma54 (σ54) is the major alternative sigma factor in E. coli and historically known for its role in nitrogen metabolism. One unique property of σ54-dependent transcription is that it recognises -12 and -24 promoter elements rather than the traditional -10 and -35 sequences. Another distinguishing feature of σ54-dependent transcription initiation is that it absolutely requires the activity of a cognate activator and ATP hydrolysis, potentially giving tight control over gene expression and a wider dynamic range than with σ70-dependent transcription. Interestingly, this activator requirement has been shown to be bypassed in vitro with σ54 mutants where the interaction with the -12 promoter DNA element is disrupted. However, σ54-dependent transcription is not readily observed in vivo for the same mutants, suggesting further barriers exist in vivo inhibiting activator independence from occurring. The -12 promoter element has been shown to be somewhat expendable for σ54 promoter binding, and activator-independent σ54 transcription would obviate the need for additional proteins and ATP. This raises the question as to why σ54-dependent transcription has not evolved towards activator independence over time, by bypassing the interaction with the -12 promoter DNA. In this study, I show that σ54-dependent inhibition of transcription was detected for several genes with σ54 binding sites in their promoter region (ytfJ, chaC, patA, argT, mdfA ybhK, and acrD). Additionally, local transcriptional repression in the RNASeq data directly correlated with proximal σ54 binding sites, suggesting a novel role for σ54 as a transcriptional repressor. Strikingly, the -12 consensus motifs were more conserved in promoters linked to σ54-dependent transcriptional inhibition, underlining the importance of this promoter element in this newly discovered putative repressive function. However, extensive screens for genes inhibiting bypass transcription in vivo failed to identify any major repressive genes that keep activator independence in check in vivo. The mechanisms maintaining activator bypass transcription at low levels in vivo still remain to be characterised.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656834 |
| Date | January 2014 |
| Creators | Schafer, Jorrit |
| Contributors | Buck, Martin |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/24804 |
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