tRNA Translocation by the Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2006
The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP͞GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 M range (37°C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G⅐GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosomecomplex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and Pi release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.
GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits
The EMBO journal, 2014
Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our resu...
[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.
BioEssays : news and reviews in molecular, cellular and developmental biology, 2014
The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms.
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.
Proceedings of the National Academy of Sciences, 2020
The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond for...
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...
Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis
Proceedings of the National Academy of Sciences, 2009
In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA·EF-Tu·GDP·kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.
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.