The <i>pcnB </i>gene encodes poly(A) polymerase I (PAP I), the major <i>Escherichia coli</i> 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 <i>pcnB</i> region was ill-defined. In this work, the <i>pcnB</i> promoter, identified by sub-cloning and primer extension analysis, was found to resemble closely a consensus s<sup>70</sup> promoter in both sequence and activity. Operon fusions of <i>pcnB </i>with <i>lacZ</i> are only slightly derepressed in a D<i>pcnB</i> background, providing little evidence of autoregulation at the level of transcription. Translation of the <i>pcnB</i> 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 <i>in vivo </i>expression of <i>pcnB</i> 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 <i>infC </i>(which encodes IF-3) can induce maximal IF-3-mediated repression of <i>pcnB</i>. Apart from the Shine-Dalgarno region, <i>pcnB</i> appears to lack any of the known sequence determinants encoded by <i>infC</i> that are believed to facilitate the use of AUU as an initiation codon. Several <i>pcnB</i> 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 <i>pcnB</i> might be subject to regulation by pyrimidine-sensitive selection of transcriptional start sites, coupled with UTP-dependent reiterative transcription. This possibility is investigated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641629 |
| Date | January 2000 |
| Creators | Binns, Nigel P. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10816 |
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