Structure-function studies of a putative ribonuclease HI from Mycobacterium tuberculosis.

Thomsen, Michelle Lesley
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Bacterial Ribonuclease HI, which ensures that initiation of DNA replication occurs at the unique oriC; locus, is encoded by rnhA. The rnhA gene from Mycobacterium smegmatis encodes a protein that is closely related to other bacterial RNases HI (Dawes et al., 1995). Activity gel analysis rletected RNase HI activity associated with proteins in whole-cell extracts of Mycobacterium tuberculosis in the 14-25 kDa size range. A putative rnhA homologue was identified in M. tuberculosis and sequence analysis revealed that the rnhA open reading frame contains an apparent fusion of two genes (Cole et al., 1998). The 5' -region of the ORF corresponds to an rnhA homologue, whereas the 3'-region contains a gene, annotated herein as pgm, which encodes a protein belonging to the phosphoglycerate mutase (POM) family of proteins. The full-length ORF, as well as the individual mhA and pgm segments, were cloned into the pMAL-c2 expression vector and recombinant proteins were overexpresssed in E. coli as maliose binding fusion proteins. Recombinant proteins were purified and rabbit polyclonal antisera raised against each one were used to probe whole cell extracts of M. tuberculosis. Cross-reaction with polypeptides of unknown identity was observed. Limited proteolysis of the recombinant proteins suggest an instability of folding in E. coli. Functional investigation of the M. tuberculosis RNase HI included complementation of an E. coli RNase HI-defective mutant, and an M. smegmatis strain carrying a defective rnhA allele integrated at its rnhA locus, with M. tuberculosis rhns-pgm supplied in trans. No complementation in either hosts was observed. Upon completion of the genome sequence of H37Rv (Cole et al., 1998), it became apparent that the rnh/i-pgm ORF is the fourth gene in an operon which includes a gene known to be involved in cobalamin biosynthesis. Significant homlogy of the PGM to CobC phosphatase of Salmonella typhimurium implicates a role for rnh/: pgm in the cobalamin biosynthetic pathway of M. tuberculosis.
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science.
Mycobacterium tuberculosis. , Tuberculosis. , Mycobacterial diseases -- Research.