Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

Abstract

AbstractPathogenic organisms encounter a broad range of stress conditions within host micro-environment and adopt variety of mechanisms to stall protein translation and protect translational machinery. Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report a growth phase-dependent conformational switch of domain III and IV helices (H54a and H67-H71) of the mycobacterium 23S rRNA. Cryo-electron microscopy (cryo-EM) structures (∼3-4 Å) of the M. smegmatis (Msm) 50S ribosomal subunit of log-phase manifested that, while H68 possesses the usual stretched conformation in one of the maps, another one exhibits an unprecedented conformation of H68 curling onto a differently oriented H69, indicating an intrinsic dynamic nature of H68. Remarkably, a 2.8Å cryo-EM map of the Msm stationary-state 50S subunit unveiled that H68 preferably acquires folded conformation in this state (closely mimicking dormant state). Formation of a bulge-out structure by H68 at the inter-subunit surface of the stationary-state 50S subunit due to the rRNA conformational changes prevents association with 30S subunit and keeps an inactive pool of the 50S subunit representing a ribosome-protection mechanism during dormancy. Evidently, this dynamic nature of H68 is an integral part of the cellular functions of mycobacterium ribosome, and irreversibly arresting H68 flexible motion would stall ribosome function. Thus, this conformational change may be exploited to develop anti-mycobacterium drug molecules.Significant statementBacteria utilize several mechanisms to reprogram the protein synthesis machinery so that their metabolism is reduced in the dormant state. Mycobacteria are capable of hiding themselves in a dormant state during physiological stresses. Our study identified a hitherto-unknown folded conformation of the helix 68 (H68) of domain IV of mycobacterial 23S rRNA, which is predominantly present in the stationary state (closely mimicking latency). Our results suggest that this conformational transition is instrumental in keeping an inactive pool of the 50S subunit in the stationary state. Irreversibly arresting such conformational dynamics would lead to protein synthesis shutdown in mycobacteria during dormancy. Thus, this folded conformation of H68 offers an excellent therapeutic intervention site to treat mycobacterial latent infection.HighlightsIdentification of a hitherto-unknown folded conformation of the helix 68 of mycobacterial 23S rRNAH68 conformation transition represents a new ribosome protection mechanism in dormant mycobacteriaThe conformational switch of mycobacterial H68 offers an excellent therapeutic intervention site