Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex

Abstract

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pre-translocation complex reveal spontaneous, large-scale conformational changes including an inter-subunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favoured within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor, cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.

Trajectories of the ribosome as a Brownian nanomachine

“…Using rigid-body fitting of domains while making use of a published cryo-EM map of the yeast ribosome (26) for reference, we observe combinations of four motions previously described in the literature and associated with the elongation cycle, as follows: (i) ratchet-like intersubunit rotation, a counterclockwise rotation of the small subunit about an axis normal to the subunit interface (Fig. 3A, solvent view) (27,28); (ii) rotation (closing movement) of the L1 stalk toward the intersubunit space (Fig. 3A, solvent and top views) (29); (iii) small subunit head swivel, a rotation of the subunit head about its long axis (Fig.…”

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

“…In the posttranslocation state, in which peptidyl-and deacyl-tRNAs occupy the P and E sites, respectively, the ribosome adopts a "nonrotated" (classical) conformation. In the pretranslocation state, a 9-12°counterclockwise rotation of the small subunit was observed for bacterial (36,42,43) and eukaryotic (44) ribosomes. This conformation of the ribosome is referred to as "rotated.…”

“…Because the IGR IRES mRNA has to undergo translocation to free its A site for alanyl-tRNA Ala , we compared the conformational states of the IRES-ribosome complexes in our structures with the known pretranslocation (rotated) and posttranslocation-like (nonrotated) tRNA-bound rabbit ribosomes (44). As in bacterial ribosome complexes, the small subunit of the pretranslocation hybrid state mammalian ribosome is rotated counterclockwise by ∼9°relative to that in the classical with the components of the ribosome (rendered as ribbons).…”

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

“…Following the formation of peptidyl-tRNA in the A-site and deacylated tRNA in the P-site as a result of the transpeptidation reaction in the LSU, the ribosome fluctuates between classical (А/А:Р/Р) and hybrid (А/А:Р/Е or А/Р:Р/Е) positions of the tRNAs (3). This pretranslocational state (PRE) is associated with a rotation of the SSU relative to the LSU (4,5). Another large-scale ribosome movement, swivelling of the SSU 'head' domain, moves the anticodon ends of the tRNAs into the ap/P:pe/E states (6).…”

“…Elongation factor 2 in complex with GTP binds to the PRE ribosome and, regardless of its initial conformation, causes an appearance of the hybrid tRNA states (5,10). After binding, the protein undergoes conformational changes that bring the second superdomain into the A-site of the SSU (11,12), where it interacts via domain IV with the decoding centre and tRNA-mRNA-complex (12)(13)(14).…”