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 withM. tuberculosisof 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 theM. tuberculosisRNase E/G homologue.To learn more about specific properties of RNase E/G homologues aM. tuberculosisRNase E/G (MycRne) was overexpressed inE. coliand purified as a 6His-tagged polypeptide.To characterise MycRne,we usedin vitrocleavage 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 toE. coliRNase E, MycRne was able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA.Although, similar toE. coliRNase 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.