Cryo-EM captures a unique conformational rearrangement in 23S rRNA helices of the Mycobacterium 50S subunit.

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 structural evidence of a hitherto unknown conformational switch of mycobacterium 23S rRNA helices (H54a and H67-H71). Cryo-electron microscopy (cryo-EM) structures (~3-4 Å) of the M. smegmatis (Msm) log-phase 50S ribosomal subunit revealed conformational variability in H67-H71 region of the 23S rRNA, and manifested that, while H68 possesses the usual stretched conformation in one class of the maps, another one exhibits a bulge-out, fused density of H68-H69 at the inter-subunit surface, indicating an intrinsic dynamics of these rRNA helices. Remarkably, altered conformation of H68 forming a more prominent bulge-out structure at the inter-subunit surface of the 50S subunit due to the conformational rearrangements of 23S rRNA H67-H71 region was clearly visualized in a 3 Å cryo-EM map of the 50S subunit obtained from the stationary phase ribosome dataset. The Msm50S subunit having such bulge-out conformation at the intersubunit surface would be incompatible for associating with the 30S subunit due to its inability to form major inter-subunit bridges. Evidently, availability of active 70S ribosome pool can be modulated by stabilizing either one of the H68 conformation.

Ribosome-membrane crosstalk: Co-translational targeting pathways of proteins across membranes in prokaryotes and eukaryotes.

Ribosomes are the molecular machine of living cells designed for decoding mRNA-encoded genetic information into protein. Being sophisticated machinery, both in design and function, the ribosome not only carries out protein synthesis, but also coordinates several other ribosome-associated cellular processes. One such process is the translocation of proteins across or into the membrane depending on their secretory or membrane-associated nature. These proteins comprise a large portion of a cell's proteome and act as key factors for cellular survival as well as several crucial functional pathways. Protein transport to extra- and intra-cytosolic compartments (across the eukaryotic endoplasmic reticulum (ER) or across the prokaryotic plasma membrane) or insertion into membranes majorly occurs through an evolutionarily conserved protein-conducting channel called translocon (eukaryotic Sec61 or prokaryotic SecYEG channels). Targeting proteins to the membrane-bound translocon may occur via post-translational or co-translational modes and it is often mediated by recognition of an N-terminal signal sequence in the newly synthesizes polypeptide chain. Co-translational translocation is coupled to protein synthesis where the ribosome-nascent chain complex (RNC) itself is targeted to the translocon. Here, in the light of recent advances in structural and functional studies, we discuss our current understanding of the mechanistic models of co-translational translocation, coordinated by the actively translating ribosomes, in prokaryotes and eukaryotes.