Seeking Species-Specific Drug Targets in Mycobacterial Gene Expression

Abstract

Gene expression in the human pathogen, Mycobacterium tuberculosis (M. tuberculosis), may possess species-specific structural features that can be targeted by drugs. Previous studies have identified, for example, several ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein) encoded by paralogous genes in the M. tuberculosis ribosome. To better understand the functions of these features and facilitate drug design, high-resolution structures are essential. Here we have used cryo-electron microscopy (cryo-EM) to solve the structures of RNA polymerase, the ribosome, and their higher order complexes. The RNA polymerase structure we have solved is a core complex consisting of all five subunits. The structure indicates a new conformation of a bridging α-helix, termed “B2”, on the β subunit. In this new conformation, the B2 helix can potentially interact with rifampicin, a firstline drug against tuberculosis, suggesting exciting potential approach to design rifampicin derivatives. For the M. tuberculosis ribosome, we have solved multiple structures. These structures reveal that, upon joining of the large and small ribosomal subunits, a 100-nucleotide long expansion segment of the M. tuberculosis 23S rRNA, named H54a or the “handle”, switches from interacting with rRNA helix H68 and rProtein uL2 to interact with rProtein bS6, forming a new intersubunit bridge “B9”. In M. tuberculosis 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. We have also characterized the highorder complexes involved in gene expression, including ribosome dimers and expressome. The biochemical and structural characterizations in this work provided a glimpse of the key components in the mycobacterial gene expression system. Most, if not all, unique features were unveiled, which should be not only further complemented with functional studies, but also taken into account for anti-tuberculosis drug development.