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Title: Aspects of the regulation of expression of pcnB, which encodes poly (A) polymerase I of Escherichia coli
Author: Binns, Nigel P.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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The pcnB gene encodes poly(A) polymerase I (PAP I), the major Escherichia coli poly(A) polymerase involved in mRNA processing, and the decay of small plasmid copy number controls RNAs. Prior to the study presented here, the transcriptional and translational organisation of the pcnB region was ill-defined. In this work, the pcnB promoter, identified by sub-cloning and primer extension analysis, was found to resemble closely a consensus s70 promoter in both sequence and activity. Operon fusions of pcnB with lacZ are only slightly derepressed in a DpcnB background, providing little evidence of autoregulation at the level of transcription. Translation of the pcnB message is shown, by site-directed mutagenesis, to commence from the ribonucleotides AUU, a triplet that is very rarely used as an initiation codon. Furthermore, it was demonstrated that at the level of translation, the in vivo expression of pcnB is specifically subject to negative regulation, over a four-fold range, by Initiation Factor-3 (IF-3), whose own translation is also initiated from an AUU codon. Additional tests showed that a single chromosomal copy of wild-type infC (which encodes IF-3) can induce maximal IF-3-mediated repression of pcnB. Apart from the Shine-Dalgarno region, pcnB appears to lack any of the known sequence determinants encoded by infC that are believed to facilitate the use of AUU as an initiation codon. Several pcnB homologues are identified from other eubacteria that potentially could utilise an AUU triplet as a translational initiation codon. A conserved run of T residues (five to seven nucleotides long) exactly nine nucleotides downstream from the -10 region of these homologues suggested that the expression of pcnB might be subject to regulation by pyrimidine-sensitive selection of transcriptional start sites, coupled with UTP-dependent reiterative transcription. This possibility is investigated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available