Chismon, David L. (2011)
Ph.D. thesis, University of Birmingham.
Restricted to Repository staff only until 01 January 2020.
This study examined the regulation of two genes, hcp and ogt, that are reported to be involved in protection from the mutagenic effects of reactive nitrogen species in Escherichia coli. Biochemical techniques were used to investigate promoter activity and the effects of the transcription factors that are reported to regulate expression of the hcp and ogt genes, namely NarL / NarP, FNR and NsrR. Transcription activation by NarL was then studied using semi-synthetic promoters. The hcp gene was found to be positively regulated by FNR and negatively regulated by NsrR. Contrary to previous reports, NarL and NarP were found to have little direct effect on expression of hcp, however, an indirect effect of NarL was detected. This study demonstrates that the indirect effect on hcp regulation of NarL is related to repression by NsrR and suggests that NarL is involved in the generation of reactive nitrogen species. The ogt gene was confirmed to be activated by NarL independently of FNR. Studies focused on characterising the different classes by which NarL / NarP can activate promoter activity independently of FNR. NarL was found to activate by class I, II and III mechanisms, whilst NarP was capable of class II activation only. Activation by NarL was studied and a library of alanine substitutions in the carboxyl terminal domain (CTD) of the α subunit of RNA polymerase was used to demonstrate direct interaction between NarL and RNA polymerase. NarL, the CTD of NarL, NarXL, NarP and the CTD of NarP were expressed from plasmids and transcription activation studied in cells lacking chromosomal narL and narP. Full-length NarL activated by Class I, II and III mechanisms, whilst the CTD of NarL only activated by class II mechanisms. Both the full-length NarP and the CTD of NarP only activated by class II mechanisms.
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