Structure of the ribosome with elongation factor G trapped in the pretranslocation state (original) (raw)

[Mechanism of tRNA translocation on the ribosome]

Molekuliarnaia biologiia

During the translocation step of the elongation cycle of peptide synthesis two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. Translocation is catalyzed by the elongation factor G (EF-G) and requires GTP hydrolysis. The fundamental biochemical features of the process were worked out in the 1970-80s, to a large part by A.S. Spirin and his colleagues. Recent results from pre-steady-state kinetic analysis and cryoelectron microscopy suggest that translocation is a multistep dynamic process that entails large-scale structural rearrangements of both ribosome and EF-G. Kinetic and thermodynamic data, together with the structural information on the conformational changes of the ribosome and of EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.

Structural basis of early translocation events on the ribosome

Nature, 2021

Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2–20 per second) and with a low error rate (around 10−3 to 10−5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expendi...

Dynamics of translation on the ribosome: molecular mechanics of translocation

FEMS Microbiology Reviews, 1999

The translocation step of protein elongation entails a large-scale rearrangement of the tRNA-mRNA-ribosome complex. Recent years have seen major advances in unraveling the mechanism of the process on the molecular level. A number of intermediate states have been defined and, in part, characterized structurally. The article reviews the recent evidence that suggests a dynamic role of the ribosome and its ligands during translocation. The focus is on dynamic aspects of tRNA movement and on the role of elongation factor G and GTP hydrolysis in translocation catalysis. The significance of structural changes of the ribosome induced by elongation factor G as well the role of ribosomal RNA are addressed. A functional model of elongation factor G as a motor protein driven by GTP hydrolysis is discussed. ß

Movement in ribosome translocation

Journal of biology, 2005

Translocation of peptidyl-tRNA and mRNA within the ribosome during protein synthesis is promoted by the elongation factor EF-G and by the hydrolysis of GTP. A new study reports that EF-G binds to ribosomes as an EF-G.GDP complex and that GTP is exchanged for GDP on the ribosome. Together with cryo-electron microscopy, this unexpected finding helps clarify the role of GTP in translocation.

Energy barriers and driving forces in tRNA translocation through the ribosome

Nature Structural & Molecular Biology, 2013

nature structural & molecular biology advance online publication a r t i c l e s Ribosomes are molecular machines that synthesize proteins from aminoacyl tRNAs, using mRNA as template. After formation of a peptide bond, the two tRNAs bound to the aminoacyl (A) and peptidyl (P) sites on the small (30S) and large (50S) ribosomal subunits translocate by more than 7 nm to the P and exit (E) sites, respectively, while the next mRNA codon moves into the A site . During translocation, tRNAs move on the 50S subunit into the hybrid A/P and P/E positions 1 with a concomitant rotation of the 30S subunit relative to the 50S subunit 2-4 . The rate-limiting step of translocation is the displacement of the codon-anticodon complexes on the 30S subunit; this, followed by the reversal of the subunit rotation, yields the post-translocation complex. Translocation is promoted by elongation factor G (EF-G) and is driven by GTP hydrolysis. In the absence of the factor, spontaneous, thermally driven tRNA translocation can occur 5-8 , and this seems to involve the same intersubunit interactions that occur in the presence of EF-G 9 . Spontaneous translocation is an equilibrium process, in which the tRNAs make rapid, spontaneous excursions in both forward and backward directions 5,6,10 . Preferential directionality is determined by the affinities of the tRNAs for their respective binding sites 5,6 . The process of translocation entails fluctuations of tRNAs 4,11-14 and of the components of the 50S subunit such as the L1 stalk 3,15-17 . A recent cryo-EM work revealed a large number of different conformational states for spontaneous, thermally driven tRNA movement through the ribosome 10 . However, precisely how the thermal fluctuations of tRNAs and of parts of the ribosome cooperatively drive the tRNA movement is unclear. It is also unclear whether and how synchronous movements-such as those involving intersubunit rotations, the L1 stalk and tRNA fMet -are coupled to one another. Furthermore, it is unknown how efficient tRNA handover from one binding site to another is achieved, despite the considerable structural changes along the translocation path. To address these questions, we combined data from X-ray crystallography and singleparticle cryo-EM with molecular dynamics (MD) simulations.

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

Nature, 2010

The elongation cycle of protein synthesis involves the delivery of aminoacyl-tRNAs to the A-site of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A-site 1,2 . The translocation reaction is catalyzed by elongation factor G (EF-G) in a GTP-dependent fashion 3 . Despite the availability of structures of various EF-Gribosome complexes, the precise mechanism by which tRNAs move through the ribosome still

The Structure of the Ribosome with Elongation Factor G Trapped in the Posttranslocational State

Science, 2009

Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with peptidyl-tRNA binding site (P-site) tRNA and mRNA shed light on various aspects of EF-G function in catalysis and translocation. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation

Molecular cell, 2003

The elongation cycle of protein synthesis is completed by translocation, a rearrangement during which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by elongation factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suggests that GTP hydrolysis drives a conformational rearrangement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.