Interaction of the G' domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-EM

During tRNA translocation on the ribosome, an arc-like connection (ALC) is formed between the G' domain of elongation factor G (EF-G) and the L7/L12-stalk base of the large ribosomal subunit in the GDP state. To delineate the boundary of EF-G within the ALC, we tagged an amino acid residue near the tip of the G' domain of EF-G with undecagold, which was then visualized with three-dimensional cryo-electron microscopy (cryo-EM). Two distinct positions for the undecagold, observed in the GTP-state and GDP-state cryo-EM maps of the ribosome bound EF-G, allowed us to determine the movement of the labeled amino acid. Molecular analyses of the cryo-EM maps show: (1) that three structural components, the N-terminal domain of ribosomal protein L11, the C-terminal domain of ribosomal protein L7/L12, and the G' domain of EF-G, participate in formation of the ALC; and (2) that both EF-G and the ribosomal protein L7/L12 undergo large conformational changes to form the ALC.     

Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

The hybrid state of tRNA binding is an authentic translation elongation intermediate

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.     

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.     

Single-molecule fluorescence measurements of ribosomal translocation dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.     

Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of the Escherichia coli ribosome. Because of the flexible nature of the region, recent X-ray crystallographic studies of the 50 S subunit failed to locate the N-terminal domain of the protein. We have determined the position of the complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with the three-dimensional map of the wild-type ribosome. Fitting of the X-ray coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis, domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the N-terminal domain to move and thereby inducing the formation of the arc-like connection with the G' domain of EF-G. The results provide a new insight into the mechanism of EF-G-dependent translocation.     

Release of ribosome-bound ribosome recycling factor by elongation factor G

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.     

Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation.     

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

Structure of the ribosome-bound cricket paralysis virus IRES RNA

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.     

Ribosome recycling factor disassembles the post-termination ribosomal complex independent of the ribosomal translocase activity of elongation factor G

Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.     

Molecular dynamics of ribosomal elongation factors G and Tu

Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region.     

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.     

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

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.     

Almost lost in translation. Cryo-EM of a dynamic macromolecular complex: the ribosome

Ribosomes are dynamic biological machines that perform numerous tasks during translation, the biosynthesis of proteins. Translocation, the movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) to progress in the reading frame of codons in the mRNA, takes place after the addition of each amino acid. This process involves large ribosome conformational changes, where tRNAs proceed through intermediate states. The structural characterization of these translocation intermediates has remained elusive. Cryo-electron microscopy (cryo-EM) produces three-dimensional averages, and translocating ribosomes poise distinct conformational states, and hence, structurally heterogeneous populations. During the last decade, the quest for visualization of translocation intermediates has progressed together with the development of classification tools in cryo-EM. Some of these new tools have recently been tested in ribosomal translocation, uncovering a clearer picture of the process. This success goes along with the latest advances in cryo-EM and illustrates how the technique offers multiple possibilities for studying macromolecular complexes engaged in dynamic reactions.     

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.     

Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Real-time evidence for EF-G-induced dynamics of helix 44 in 16S rRNA

The penultimate stem-loop of 16S ribosomal RNA (rRNA), helix 44, plays a central role in ribosome function. Using time-resolved dimethyl sulfate (DMS) probing, we have analyzed time-dependent modifications that occur at specific bases in this helix near the decoding region, resulting from the binding of elongation factor G (EF-G) in various forms. When EF-G-GTP is bound to 70S ribosomes, bases A1492 and A1493 are immediately protected, while other bases in the region show either no change or enhanced modification. When apo-EF-G is bound to 70S ribosomes and GTP is added, substantial transient time-dependent enhancement occurs at bases A1492 and A1493, with somewhat less enhancement occurring at base A1483, all in the first 45 ms. When mRNA and deacylated tRNAs are bound to the 70S ribosome and EF-G-GTP is added, bases A1492 and A1493 again show substantial and continued enhancement, while bases A1408, A1413, and A1418 all show time-dependent protection. These results provide primary, real-time evidence that EF-G induces direct or indirect structural changes in this region as EF-G is bound and as GTP is hydrolyzed.     

The structural changes in the ribosome during the elongation cycle

The plenty of data about structural changes in the ribosome during its functioning has been accumulated. The most interesting information on such changes was obtained by cryo-EM of various ribosomal complexes with the ligands and by combination of rRNA site-directed mutagenesis with the analysis of structural changes in ribosome by chemical modification technique (chemical probing). The most studied structural transformations of the ribosome interacting with tRNAs and elongation factors are considered in this review. The structural rearrangements are discussed in the context of interactions between the functional centers of the ribosome. We also describe the system of tertiary contacts between the rRNA helices and proteins which forms the universal structure in the ribosome. We pay attention that by means of such system the allosteric conformational signal can be transmitted between the functional centers. Besides the discussion of different biochemical data in the scope of structural data we also consider the hypothesis that the position of GTPase associated center (GAC) in the ribosome regulates the binding of elongation factors.