Dullaghan, Edith Mary; (2000) Analysis of gene regulation by Mycobacterium tuberculosis Lexa. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this project is to analyse the regulation of gene expression by Mycobacterium tuberculosis LexA protein. The response to DNA damage by bacteria is highly conserved, involving the co-ordinated expression of more than 20 genes. The key regulatory elements are the RecA and LexA proteins, the activator and repressor respectively. Li addition to understanding the DNA binding properties of LexA, a detailed definition of the LexA binding site will allow the identification of other LexA regulated genes from the recently completed M. tuberculosis genome sequence. Under normal conditions LexA binds to a specific DNA sequence, the SOS box upstream of the genes it regulates and represses transcription. M tuberculosis LexA protein has been shown to bind to the same motif as that found upstream of DNA damage-inducible genes in Bacillus subtilis. The consensus sequence (GAAC-N4-GTTC) has been proposed to function as an operator site that is required for regulation of the SOS system of B. subtilis. Following the discovery of a perfect consensus motif which failed to bind LexA, the effects on LexA binding of various changes to the bases flanking the core motif from upstream of M tuberculosis recA were assessed. The results showed that the sequence determining LexA binding in M tuberculosis is more extensive than the original defined binding site of B. subtilis. These mutations were also introduced into the recA promoter linked to a lacZ reporter gene to assess their effects on gene expression in vivo. In order to investigate the core motif (GAAC- N4-GTTC) changes were made to each base in one half-site individually and the effects of these changes on LexA binding were assessed by gel retardation. To aid interpretation of the binding studies the intracellular concentration of LexA molecules was determined in M. tuberculosis H37Rv. Interestingly, upstream of the M. tuberculosis LexA coding sequence there are three motifs similar to the B. subtilis consensus sequence (GAAC-N4-GTTC), one of which is atypical (GAAC-N4-GTTT, GAAC-N4-GATC and ACTC-N4- GTTC). All three motifs were shown to bind purified M. tuberculosis LexA, but with very different affinities when in isolation. Footprinting analysis of LexA binding to a fragment containing all three sites was undertaken to examine the order of binding and any cooperativity. The presence of multiple binding sites for LexA might allow a more subtle level of regulation than would be possible with a single site. The roles of the three sites were investigated by constructing mutants with individual, or pairs of, or all three boxes knocked out in a fusion of this region to the reporter gene lacZ. Footprinting experiments were undertaken to confirm binding was eliminated where expected and to examine any effect on binding at the other sites. Concurrently, assays of the reporter gene were used to examine the effect of these mutations on lexA expression in response to DNA damage. These analyses revealed a complex interplay amongst the binding sites.

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