Cochrane, S.K.T.; (2004) Antitermination in Mycobacterium tuberculosis. Doctoral thesis , University of London.

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Abstract

Mycobacterium tuberculosis is the leading cause of death from a single infectious agent. The varying efficacy of the BCG vaccine and the emergence of multi-drug resistant strains of M. tuberculosis have made it essential that novel drug and vaccine targets are identified. The antitermination mechanism, probably used in transcriptional regulation of the single rrn operon, is one such target. Antitermination is a mechanism by which RNA polymerase is able to transcribe through both Rho-dependent and-independent terminators. Antitermination in association with Nus (N-utilising) factors was initially discovered within the ?-phage where it regulates the transcription of early and late genes. Subsequent investigations in Escherichia coli demonstrated antitermination and the Nus proteins were involved in transcriptional regulation of the seven rrn operons present in the genome. The presence of Nus homologs and Nut sequences (the assembly sites for the antitermination complex) within the M. tuberculosis genome indicate that antitermination may occur during rrn operon transcription. The aim of this project has thus been an understanding of the characteristics, functions and interactions of the Nus proteins within the M. tuberculosis antitermination complex. A definite, yet likely to be weak, interaction between NusB and NusE is shown. The stoichiometry of this interaction (NusB monomer or dimer bound to NusE monomer) was investigated and results may indicate a heterodimer. The dissociation equilibrium constant for the NusB dimer was estimated to be 8 x 10-8 M. The NusG protein was characterised and shown to be monomeric in solution with a highly elongated shape with approximately 20% ?-helical secondary structure. A C-terminal domain, containing the KOW rRNA binding motif, was identified by limited proteolysis. Lastly the Rho termination factor was over-expressed and purified and interactions with NusA and NusG investigated. No interactions have yet been detected.

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