The process of mRNA–tRNA translocation

Abstract

In the elongation cycle of translation, translocation is the process that advances the mRNA-tRNA moiety on the ribosome, to allow the next codon to move into the decoding center. New results obtained by cryoelectron microscopy, interpreted in the light of x-ray structures and kinetic data, allow us to develop a model of the molecular events during translocation.protein synthesis ͉ ribosome ͉ translation ͉ EF-G ͉ cryo-EM S ynthesis of proteins from their building blocks, the amino acids, is a fundamental process in the cells of all living organisms, be it animal, plant, or bacteria. The discovery that the macromolecular assembly that facilitates this process, the ribosome, is highly conserved in all essential parts has lent additional credence to the idea of the unity of all life at the molecular level.The ribosome is a very large (2.4 MDa in eubacteria) ribonucleicprotein complex composed of two distinct subunits, the small subunit (30S) charged with the task of decoding the genetic message carried by the messenger RNA (mRNA), the large subunit (50S) to the catalysis of peptide bond formation. Instrumental for these fundamental processes is the interaction of the ribosome with transfer RNA (tRNA), a small L-shaped molecule that embodies in its various forms the association of each amino acid with a threebase ''word'' of the genetic code, the codon. Translation is based on the mutual recognition, by partial Watson-Crick pairing, between the codon on the mRNA and the anticodon of the tRNA carrying the corresponding amino acid. In facilitating tRNA selection, decoding, and the stepwise formation of the polypeptide, ribosomal RNA (rRNA) acts as both a structural framework and a catalyst.Despite the success in the elucidation of ribosomal structure by x-ray crystallography, the detailed mechanism by which translation of mRNA code into peptide proceeds is still only scantly understood. One of the obstacles we face is that although the process is complex and dynamic, x-ray crystallography represents the molecule in a static form-packed in a crystal, moreover, whose very stability depends on intermolecular contacts that are largely nonphysiological. Of crucial importance for the understanding of the multistep translation process is the knowledge of how the ribosome interacts with its ligands, notably (apart from the most crucial ligands mRNA and tRNAs) the various factors catalyzing initiation, elongation, termination, and recycling. To date, with the exception of ribosomal complexes containing eubacterial release (RF1 or RF2) (1) or recycling (RRF) (2, 3) factors, there exists no x-ray structure of a factor-ribosome complex. The crystal structures of individual subunits complexed with initiation (4-6) and recycling (7) factors have also been solved; however, crystallographic data of elongation factors bound to the ribosome are currently still not available. Moreover, to date, despite many efforts, no atomic structure is available for a eukaryotic ribosome.Increasingly, within the past decade, cryo-electron microscopy (...

A Central Interdomain Protein Joint in Elongation Factor G Regulates Antibiotic Sensitivity, GTP Hydrolysis, and Ribosome Translocation

“…Domains III-V of EF-G undergo large rotational motions, which become progressively amplified from switch II to the tip of domain IV (11,25,36). In the ribosome, the 30 S and 50 S subunits rotate relative to each other, and the head of the 30 S subunit also rotates, which ultimately moves the mRNA and two tRNAs in the ribosome (37).…”

Simulating movement of tRNA through the ribosome during hybrid-state formation

“…The ribosome undergoes a range of complex conformational rearrangements during its functional cycle. [19][20][21][22] In order for the ribosome to read mRNA and synthesize new proteins, it recruits tRNA molecules. On the ribosome, there are three tRNA binding sites (A, P, and E) on the small (30S) and large (50S) subunits, where each tRNA molecule sequentially transits all three sites.…”

The translation elongation cycle—capturing multiple states by cryo-electron microscopy

“…In discussing the process of mRNA-tRNA translocation following peptidyl transfer, it is useful to distinguish two stages [28,29]: in the first stage, which is reversible, A-and P-site tRNAs are advanced on the large subunit as the small subunit rotates, while their anticodon stem-loops remain firmly anchored on the small subunit along with the mRNA. The sites assumed by the tRNAs intermittently, A/P and P/E, were discovered by Moazed & Noller [30], and are called hybrid sites.…”

“…Intersubunit rotation is a 'natural' motion of the PRE ribosome in thermal equilibrium, based on the unique bipartite architecture of this molecule [6,28,61]. Examination of the binding interactions between the two subunits, as revealed by X-ray and cryo-EM structures (see latest tabulation by Liu & Fredrick [76]) yields some clues on the distribution of rotation states as well as the molecular mechanism for the fine-grained control of motion.…”