Towards new antituberculotic targets: biochemical characterisation of mycobacterial RNase E/G

Abstract

The World Health Organization estimates that each year 3 million people die from tuberculosis (TB) and 8 million people become infected. No new anti-TB drugs have been introduced in the past 30 years, even though their development becomes increasingly important to face new challenges posed by multidrug-resistant and extensively drug-resistant strains and by acute infection with <i>M. tuberculosis</i> of HIV positive patients. Owing to its apparently important role in RNA metabolism, the RNase E/G family of endoribonucleases can be considered as a promising target for antimicrobial drugs. This consideration promted us to characterise biochemical properties of the <i>M. tuberculosis</i> RNase E/G homologue. To learn more about specific properties of RNase E/G homologues a <i>M. tuberculosis</i> RNase E/G (MycRne) was overexpressed in <i>E. coli</i> and purified as a 6His-tagged polypeptide<i>.</i> To characterise MycRne<i>,</i> we used <i>in vitro</i> cleavage assays and primer extension analysis of total RNA extracted from mycobacteria. We show that affinity purified MycRne has an endoribonucleolytic activity, which is dependent on the 5′-phosphorylation status of RNA. We could also show that RNase E/G has Mg²⁺ dependent activity and similar to <i>E. coli</i> RNase E, MycRne was able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA. Although, similar to <i>E. coli</i> RNase E, the mycobacterial RNase E/G homologue plays a role in rRNA processing, the substrate specificities of these enzymes show differences. This suggests that RNase E/G can be used as a promising target for antimicrobial drugs that can be optimized to specifically target pathogenic species.