Molecular and genetic analysis of RepA from the P307 RepFIB replicon

Abstract

The work in this Thesis concerns the replication control system of the P307 plasmid RepFIB replicon. The basic replicon occupies ≈ 1.6kb of DNA and contains a single large open reading frame (repA) flanked on either side by a series DNA repeat elements. The organisational structure of fie replicon has placed RepFIB into the Step function class of replicons. The placement of RepFIB within this group, as well as a strong homology between RepFIB and mini-P1, has resulted in a series of predictions concerning the control elements of RepFIB replication. The aim of this work was to test some of these predictions and to characterise the fundamental control elements utilised by the replicon. This Thesis describes three different active promoter elements found embedded within the repeat elements flanking repA. Although the functional significance of two of the promoter is unknown (orip and EFp), the third is responsible for the expression of RepA and has been designated 'repAp'. All three promoters are sensitive to RepA in trans, demonstrating that repA is autoregulated and that RepA is a DNA-binding protein capable of recognising copies of the repeat elements. RepA DNA-binding has also been demonstrated in vitro using a modification of the Western analysis technique (referred to a 'Western-DNA') in order to complement the in vivo experimental results. Although the coding region of repA had been determined in earlier work, the identification of the translational start codon was uncertain. This uncertainty has been resolved by limited N-terminal sequence analysis of a RepA:β-galactosidase fusion protein which has demonstrated that translation begins from a CTG codon located upstream of the predicted start sites. Finally, a series of genetic experiments have been used to determine the functional significance of RepA binding to the repeat elements. The repeat group upstream of repA are involved in autoregulation and also form part of the origin of replication, whilst the downstream repeats appear to be involved in the sensing and setting of plasmid copy number. Although the work presented in this Thesis does not directly test the applicability for RepFIB of various control models proposed to explain the behaviour of Step function replicons, the nature and type of control elements identified in RepFIB support the placement of RepFIB within the Step function class. As a result of this work, it is clear that RepFIB is confronted by the same kind of control paradox faced by replicons such as mini-P1 and mini-F. All three replicons use autoregulation and titration to control the supply of initiator protein required for replication. However, concurrent autoregulation and titration appear to be incompatible in current control models, and the identification of both mechanisms in these replicons has lead to a control paradox. Some of the results presented here suggest potentially valuable avenues of future research which may help resolve the paradox faced by RepFIB and other Step function replicons.