Sigma-70 is the subunit of <i>Escherichia coli</i> RNA polymerase which confers specificity for initiation of transcription at most promoters in actively growing cells. The protein shares four conserved regions with major and many alternative sigmas within all studied prokaryotes. However, a stretch of 245 amino acids lying between conserved regions 1 and 2, is absent from all minor sigmas and even from the similar and functionally homologous sigma-43 of <i>Bacillus subtilis</i>. Deletion of these 245 residues from sigma-70 generates a protein which retains significant <i>in vitro</i> and partial <i>in vivo</i> function. The three natural cysteine residues of sigma-70 are confined to the deleted region. Plasmids allowing overproduction of the internally deleted protein and of a number of single-cysteine substituted mutant derivatives were constructed, and these were tested for function <i>in vivo</i> using a chimaeric sigma/promoter system. The overproduced proteins were isolated employing a novel purification method, and their activities analysed <i>in vitro</i> at l<sub>PR</sub> and T7A1 promoters. An extraordinary tendency towards reversible aggregation and hydrophobicity were observed. A possible role of the 245 amino acids domain is discussed. The <i>scrP</i> gene encodes sigma cross-reacting protein, originally identified during a search for alternative sigma factors, but was later shown not to be a sigma protein. As part of the effort to elucidate its possible function, the regulation of expression of the <i>scrP</i> gene was studied. Transcriptional fusions of <i>lacZ</i> to <i>scrP</i> and <i>arcB</i> and <i>mtqA</i>, the genes adjacent to it, were produced and utilised to analyse the expression of ScrP. <i>scrP</i> belongs to the same operon as <i>arcB</i> and <i>mtgA</i>, and is transcribed both from its own promoter and from the promoter upstream from <i>arcB</i>. Further analysis was used to demonstrate that ScrP is localised in the periplasm of <i>E. coli</i>.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:653218 |
| Date | January 1999 |
| Creators | Kasciukovic, Taciana |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12329 |
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