Identification of two distinct hybrid state intermediates on the ribosome

High spatial and time resolution single-molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the aminoacyl (A) and peptidyl (P) sites of the ribosome. Our investigation of tRNA motions, quantified on wild-type, mutant, and L1-depleted ribosome complexes, reveals a dynamic exchange between three metastable tRNA configurations, one of which is a previously unidentified hybrid state in which only deacylated-tRNA adopts its hybrid (P/E) configuration. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements of the ribosome particle.     

Capturing Transition Paths and Transition States for Conformational Rearrangements in the Ribosome

To reveal the molecular determinants of biological function, one seeks to characterize the interactions that are formed in conformational and chemical transition states. In other words, what interactions govern the molecule’s energy landscape? To accomplish this, it is necessary to determine which degrees of freedom can unambiguously identify each transition state. Here, we perform simulations of large-scale aminoacyl-transfer RNA (aa-tRNA) rearrangements during accommodation on the ribosome and project the dynamics along experimentally accessible atomic distances. From this analysis, we obtain evidence for which coordinates capture the correct number of barrier-crossing events and accurately indicate when the aa-tRNA is on a transition path. Although a commonly used coordinate in single-molecule experiments performs poorly, this study implicates alternative coordinates along which rearrangements are accurately described as diffusive movements across a one-dimensional free-energy profile. From this, we provide the theoretical foundation required for single-molecule techniques to uncover the energy landscape governing aa-tRNA selection by the ribosome.     

Mechanism of tRNA Translocation on the Ribosome

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 in the ribosome and EF-G, provide a detailed mechanistic model of translocation and suggest a mechanism of translocation catalysis by EF-G.     

Mechanism of tRNA translocation on the ribosome

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.     

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.     

The ribosome as an allosterically regulated molecular machine

The ribosome as a complex molecular machine undergoes significant conformational rearrangements during the synthesis of polypeptide chains of proteins. In this review, information obtained using various experimental methods on the internal consistency of such rearrangements is discussed. It is demonstrated that allosteric regulation involves all the main stages of the operation of the ribosome and connects functional elements remote by tens and even hundreds of angstroms. Data obtained using Förster resonance energy transfer (FRET) show that translocation is controlled in general by internal mechanisms of the ribosome, and not by the position of the ligands. Chemical probing data revealed the relationship of such remote sites as the decoding, peptidyl transferase, and GTPase centers of the ribosome. Nevertheless, despite the large amount of experimental data accumulated to date, many details and mechanisms of these phenomena are still not under- stood. Analysis of these data demonstrates that the development of new approaches is necessary for deciphering the mech- anisms of allosteric regulation of the operation of the ribosome.     

Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA^Trp miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.     

Contribution of intersubunit bridges to the energy barrier of ribosomal translocation

In every round of translation elongation, EF-G catalyzes translocation, the movement of tRNAs (and paired codons) to their adjacent binding sites in the ribosome. Previous kinetic studies have shown that the rate of tRNA–mRNA movement is limited by a conformational change in the ribosome termed ‘unlocking’. Although structural studies offer some clues as to what unlocking might entail, the molecular basis of this conformational change remains an open question. In this study, the contribution of intersubunit bridges to the energy barrier of translocation was systematically investigated. Unlike those targeting B2a and B3, mutations that disrupt bridges B1a, B4, B7a and B8 increased the maximal rate of both forward (EF-G dependent) and reverse (spontaneous) translocation. As bridge B1a is predicted to constrain 30S head movement and B4, B7a and B8 are predicted to constrain intersubunit rotation, these data provide evidence that formation of the unlocked (transition) state involves both 30S head movement and intersubunit rotation.     

Exploring the helix-coil transition via all-atom equilibrium ensemble simulations

The ensemble folding of two 21-residue alpha-helical peptides has been studied using all-atom simulations under several variants of the AMBER potential in explicit solvent using a global distributed computing network. Our extensive sampling, orders of magnitude greater than the experimental folding time, results in complete convergence to ensemble equilibrium. This allows for a quantitative assessment of these potentials, including a new variant of the AMBER-99 force field, denoted AMBER-99 phi, which shows improved agreement with experimental kinetic and thermodynamic measurements. From bulk analysis of the simulated AMBER-99 phi equilibrium, we find that the folding landscape is pseudo-two-state, with complexity arising from the broad, shallow character of the "native" and "unfolded" regions of the phase space. Each of these macrostates allows for configurational diffusion among a diverse ensemble of conformational microstates with greatly varying helical content and molecular size. Indeed, the observed structural dynamics are better represented as a conformational diffusion than as a simple exponential process, and equilibrium transition rates spanning several orders of magnitude are reported. After multiple nucleation steps, on average, helix formation proceeds via a kinetic "alignment" phase in which two or more short, low-entropy helical segments form a more ideal, single-helix structure.     

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.     

Ligand-Induced Changes of the Apparent Transition-State Position in?Mechanical Protein Unfolding

Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx^‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx^‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx^‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins.     

Insights into the molecular determinants of EF-G catalyzed translocation

Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 3'-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 3'-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.     

Assembly of the 30S ribosomal subunit

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.     

Diffusion control in biochemical specificity

Biochemical specificity is critical in enzyme function, evolution, and engineering. Here we employ an established kinetic model to dissect the effects of reactant geometry and diffusion on product formation speed and accuracy in the presence of cognate (correct) and near-cognate (incorrect) substrates. Using this steady-state model for spherical geometries, we find that, for distinct kinetic regimes, the speed and accuracy of the reactions are optimized on different regions of the geometric landscape. From this model we deduce that accuracy can be strongly dependent on reactant geometric properties even for chemically limited reactions. Notably, substrates with a specific geometry and reactivity can be discriminated by the enzyme with higher efficacy than others through purely diffusive effects. For similar cognate and near-cognate substrate geometries (as is the case for polymerases or the ribosome), we observe that speed and accuracy are maximized in opposing regions of the geometric landscape. We also show that, in relevant environments, diffusive effects on accuracy can be substantial even far from extreme kinetic conditions. Finally, we find how reactant chemical discrimination and diffusion can be related to simultaneously optimize steady-state flux and accuracy. These results highlight how diffusion and geometry can be employed to enhance reaction speed and discrimination, and similarly how they impose fundamental restraints on these quantities.     

Ribosomal translocation: EF-G turns the crank

Recent results from cryo-electron microscopy have shown that substantial structural rearrangements in both elongation factor EF-G and the ribosome occur during tRNA translocation. The observed sites of interaction between EF-G and the ribosome are consistent with molecular mimicry models for EF-G function.     

Mapping folding energy landscapes with theory and experiment

The detailed characterization of the overall free energy landscape associated with the folding process of a protein is the ultimate goal in protein folding studies. Modern experimental techniques and all-atom simulations provide a way to obtain accurate thermodynamic and kinetic measurements, but they are oftentimes restricted to probe limited regions of a protein landscape. Although simplified protein models can access larger regions of the landscape, they are built on assumptions and approximations that can affect the accuracy of the results. We review here recent promising approaches that allow to combine the complementary strengths of theory and experiment for a more complete characterization of a protein folding landscape at multiple resolutions. Recent results and possible applications are discussed.     

The mechanics of translocation: a molecular "spring-and-ratchet" system

The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a "spring-and-ratchet" system within the translocation process.     

Optimal combination of theory and experiment for the characterization of the protein folding landscape of S6: how far can a minimalist model go?

The detailed characterization of the overall free energy landscape associated with the folding process of a protein is the ultimate goal in protein folding studies. Modern experimental techniques provide accurate thermodynamic and kinetic measurements on restricted regions of a protein landscape. Although simplified protein models can access larger regions of the landscape, they are oftentimes built on assumptions and approximations that affect the accuracy of the results. We present a new methodology that allows to combine the complementary strengths of theory and experiment for a more complete characterization of a protein folding landscape. We prove that this new procedure allows a simplified protein model to reproduce remarkably well (correlation coefficient > 0.9) all experimental data available on free energies differences upon single mutations for S6 ribosomal protein and two circular permutants. Our results confirm and quantify the hypothesis, recently formulated on the basis of experimental data, that the folding landscape of protein S6 is strongly affected by an atypical distribution of contact energies.     

Deletion of a conserved, central ribosomal intersubunit RNA bridge

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.     

GTP hydrolysis by EF‐G synchronizes tRNA movement on small and large ribosomal subunits

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 results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.