The Gene cloning and biochemical and genetic analysis of the ribonuclease HI from Mycobacterium smegmatis

Abstract

Two members of the genus Mycobacteria are the causative agents of tuberculosis and leprosy, and many others are opportunistic pathogens in immune-compromised individuals. An alarming :resurgence of tuberculosis in First World countries, coupled with the high toll of mortality that this disease exacts in the Developing World, has led the World Health Organisation to declare tuberculosis a global emergency. Relatively little is known, however, about the molecular biology of the mycobacteria Elucidation of mechanisms involved in the regulation of 'fundamental processes in this genus, such as DNA replication, are expected to provide a basis for understanding mechanisms of pathogenesis in the mycobacteria. RNase Hl, encoded by rnhA, plays a central role m DNA replication in Escherichia coli by fixing initiation of DNA replication to one origin. The presence of a similar activity in the mycobacteria was therefore investigated by probing a genetically manipulable. member of the mycobacteria, Ai. smegmatis, for a homologue of the mhA gene. Degenerate primers based on blocks of amino acids conserved in other bacterial RNase HI homologues had been shown to amplify an internal portion ofmhA fromM. smegmatis (Mizrahi et al., 1993). This peR generated probe was used in this study to isolate the entire gene from M smegma tis, which was cloned as two overlapping fragments and sequenced. The deduced amino acid sequence ofM smegmatis rnhA coded for an RN~"e HI of 159 amino adds which showed 50% identity to E. coli RNase HI. A recombinant form afM smegma/is RNase III was expressed as part of a fusion protein to the maltose binding protein to facilitate in vitro biochemical characterisation of the protein. The recombinant RNase fIT exhibited hydrolytic activity specific for the RNA strand of an RNADNA hybrid and generated a similar distribution of hydrolysis products to that of E. coli RNase Hl,