Pretranslocation Research Articles

Structure of the translating Neurospora ribosome arrested by cycloheximide

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

Bayesian-Estimated Hierarchical HMMs Enable Robust Analysis of Single-Molecule Kinetic Heterogeneity

Single-molecule kinetic experiments allow the reaction trajectories of individual biomolecules to be directly observed, eliminating the effects of population averaging and providing a powerful approach for elucidating the kinetic mechanisms of biomolecular processes. A major challenge to the analysis and interpretation of these experiments, however, is the kinetic heterogeneity that almost universally complicates the recorded single-molecule signal versus time trajectories (i.e., signal trajectories). Such heterogeneity manifests as changes and/or differences in the transition rates that are observed within individual signal trajectories or across a population of signal trajectories. Because characterizing kinetic heterogeneity can provide critical mechanistic information, we have developed a computational method that effectively and comprehensively enables such analysis. To this end, we have developed a computational algorithm and software program, hFRET, that uses the variational approximation for Bayesian inference to estimate the parameters of a hierarchical hidden Markov model, thereby enabling robust identification and characterization of kinetic heterogeneity. Using simulated signal trajectories, we demonstrate the ability of hFRET to accurately and precisely characterize kinetic heterogeneity. In addition, we use hFRET to analyze experimentally recorded signal trajectories reporting on the conformational dynamics of ribosomal pre-translocation (PRE) complexes. The results of our analyses demonstrate that PRE complexes exhibit kinetic heterogeneity, reveal the physical origins of this heterogeneity, and allow us to expand the current model of PRE complex dynamics. The methods described here can be applied to signal trajectories generated using any type of signal and can be easily extended to the analysis of signal trajectories exhibiting more complex kinetic behaviors. Moreover, variations of our approach can be easily developed to integrate kinetic data obtained from different experimental constructs and/or from molecular dynamics simulations of a biomolecule of interest.

Single-molecule FRET studies on interactions between elongation factor 4 (LepA) and ribosomes

Elongation factor 4 (EF4) is one of the highly conserved translational GTPases, whose functions are largely unknown. Structures of EF4 bound ribosomal PRE-translocation and POST-translocation complexes have both been visualized. On top of cellular, structural, and biochemical studies, several controversial models have been raised to rationalize functions of EF4. However, how EF4 modulates elongation through its interactions with ribosomes has not been revealed. Here, using single-molecule fluorescence resonance energy transfer assays, we directly captured short-lived EF4·GTP bound ribosomal PRE and POST translocation complexes, which may adopt slightly different conformations from structures prepared using GDP, GDPNP, or GDPCP. Furthermore, we revealed that EF4·GTP severely impairs delivery of aminoacyl-tRNA into the A-site of the ribosome and moderately accelerates translocation. We proposed that functions of EF4 are to slow overall elongation and to stall majority of ribosomes in POST states under stress conditions.

Elongation factor G initiates translocation through a power stroke

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways.

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Rotational Motions of Domains in Elongation Factor G Detected by Single-Molecule Polarized Fluorescence Microscopy

During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule polarized total internal reflection fluorescence (polTIRF) microscopy to characterize tilting and rotational fluctuations within specific domains of EF-G. When EF-G binds to the ribosomal pre-translocation (PRE) complex, domains I and IV of EF-G undergo small rotations (10-15°) in conjunction with translocation, whereas domain III shows a much greater angular change, averaging 50°. Viomycin (Vio), which prevents translocation, reduces the rotational motions of domain III to 10-15° but has virtually no effect on the other domains. Spectinomycin also reduces domain III motions but less strongly than Vio. EF-G binding to ribosomal initiation complexes lacking A-site tRNA gives a similar pattern of domain rotations, but with shorter dwell times. In this case, the large rotation of domain III is barely inhibited by Vio. Irrespective of completion of translocation or presence of A-site tRNA, the initial 10-15° rotations of EF-G domains I, III and IV in the ribosome/EF-G complex indicate that the EF-G initially shifts the minimum of the free energy profile in the direction of translocation, suggesting that EF-G generates a force on the ribosome and/or the mRNA and tRNAs. Near the end of translocation, domain III completes its rotation either to push the mRNA and tRNAs (a working stroke) or to prevent reversal of translocation driven by thermal fluctuations (a ratchet). Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

Monitoring Translation with Modified mRNAs Strategically Labeled with Isomorphic Fluorescent Guanosine Mimetics

Here we examine three mRNAs, site-specifically modified at codon positions 4, 5, and 6 with a new emissive and responsive isosteric guanosine mimetic ((th)G), with the goal of developing real time assays for monitoring translation-related events at nucleotide resolution. All three emissive mRNAs tested form initiation (70SIC), pretranslocation (PRE), and posttranslocation (POST) complexes. In most cases spectral differences are seen on binding of the mRNA to the ribosome during 70SIC formation and on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes. These differences allow measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Such measurements directly identify a specific step during PRE complex formation, provisionally assigned to codon:anticodon-loop base pair formation, that follows aa-tRNA.EF-Tu.GTP ternary complex binding to the ribosome and precedes aa-tRNA accommodation into the A-site of the ribosome. These observations demonstrate not only the functionality of mRNAs modified with the emissive guanosine mimetic but also the potential this mimetic offers for observing the formation and disappearance of discrete intermediates during the polypeptide elongation cycle.

Monitoring Translation with Modified MRNAs Strategically Labeled with Isomorphic Fluorescent Nucleosides

To kinetically monitor translation-related events at a nucleotide resolution, a series of mRNAs, site-specifically modified at key codon positions with new emissive and responsive isomorphic nucleotides (thC, thU, thG - work withthA is commencing) have been studied. mRNAs modified with thC, thU and thG (excluding thU2 in the start codon) form an initiation complex (70SIC) and show significant spectral changes between the free and 70SIC-bound fluorescent mRNAs. All emissive mRNAs tested containing labeled nucleotides in the second codon facilitate aa-tRNA A-site binding [as part of an aa-tRNA.EF-Tu.GTP ternary complex (TC)] and pretranslocation (PRE) complex formation, although with somewhat different relative efficiencies, and all PRE complexes can be translocated via addition of EF-G.GTP to form postranslocation (POST) complexes. In most cases spectral differences are seen on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes, allowing measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Preliminary comparison of tRNA A-site binding kinetics, as monitored by the fluorescent mRNAs, with other measures of PRE complex formation indicate that TC binding to the ribosome is faster than codon-anticodon interaction, which in turn is somewhat faster than tRNA accommodation. These preliminary observations not only show excellent performance of mRNAs modified with our emissive RNA alphabet, but also clearly demonstrate their potential to illuminate the formation and disappearance of discrete intermediates in the polypeptide elongation cycle.

Intermediate states during mRNA–tRNA translocation

Recent studies support the notion that the pre-translocation (PRE) ribosomal complex functions, at least in part, as a Brownian machine, stochastically fluctuating among multiple conformations and transfer RNA (tRNA) binding configurations. Apart from the relatively more energetically stable conformational states of the PRE complex, termed macrostate I (MS I) and macrostate II (MS II), several additional intermediate states have been recently discovered. Structural and kinetic analyses of these states, made possible by cryogenic-electron microscopy (cryo-EM), X-ray crystallography, and single-molecule fluorescence resonance energy transfer (smFRET), have provided important insights into the translocation process, which is now understood to proceed, at least in the first step of the process, as a Brownian machine that is transiently stabilized in the 'productive' MS II conformation by the binding of the translocase elongation factor G (EF-G).

The Magnesium Dependence of Ribosome and tRNA Dynamics in Single Pre-Translocation Ribosomal Complexes

The structural dynamics of the cellular translational machinery are essential to the mechanism of protein synthesis. During the elongation stage of protein synthesis, for example, the translating ribosome oscillates between two functionally important global conformational states, referred to as GS1 and GS2, as it translocates along its messanger RNA (mRNA) template and sequentially incorporates aminoacylated transfer RNA (tRNA) substrates in the order specified by the mRNA. Structural transitions of the pre-translocation (PRE) ribosomal complex between GS1 and GS2 encompass extensive remodeling of ribosome-ribosome, ribosome-tRNA, and ribosome-mRNA interactions and regulate movement of the mRNA and its associated tRNAs through the ribosome by precisely one codon. Motivated by the crucial role that Mg2+ ions play in stabilizing RNA-RNA interactions and by the known Mg2+ dependence of the translocation reaction, we have used total internal reflection fluorescence microscopy to perform single-molecule fluorescence resonance energy transfer experiments on fluorescently labeled PRE complexes as a function of Mg2+ concentration. The data reveal how the thermodynamic stabilities of GS1 and GS2 and, consequently, the rates of GS1â†'GS2 and GS2â†'GS1 transitions in PRE complexes depend on the concentration of Mg2+ ions. Interpreting our data within the context provided by the atomic-resolution structures of GS1- and GS2-like ribosomal complexes and the well-characterized Mg2+ dependence of the translocation reaction rate will enable us to develop a comprehensive understanding of the way in which specific Mg2+ binding sites affect PRE complex conformational dynamics that are critical for the translocation step of translation elongation.

The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis

EF4 (LepA), a strongly conserved protein, is important for bacterial growth and functional protein biosynthesis under certain conditions and is quite similar structurally to the translocase EF-G. The elongation cycle in protein synthesis is characterized by ribosome oscillation between pretranslocation (PRE) and posttranslocation (POST) complexes. Here, using ensemble single turnover and equilibrium experiments, as well as single molecule FRET measurements, we demonstrate that EF4 can compete with EF-G for binding to the PRE complex. Such EF4 binding results in formation of a complex, denoted X(3), that effectively sequesters a catalytically active ribosome, leading to a transient inhibition of elongation that provides a mechanism for optimization of functional protein synthesis. Earlier [Liu H, et al. (2010) J Mol Biol 396:1043-1052] we demonstrated that EF4 also reacts with POST complex, leading to the formation of a complex, I(3), that appears to be identical with X(3). Our present results strongly suggest that PRE complex is the principal target of EF4 action on translation, rather than POST complex as had been previously supposed.

Transfer RNA–mediated regulation of ribosome dynamics during protein synthesis

Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during 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.

A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution.

We present a microfluidic approach for single-molecule studies of the temperature-dependent behavior of biomolecules, using a platform that combines microfluidic sample handling, on-chip temperature control, and total internal reflection fluorescence (TIRF) microscopy of surface-immobilized biomolecules. With efficient, rapid, and uniform heating by microheaters and in situ temperature measurements within a microfluidic flowcell by micro temperature sensors, closed-loop, accurate temperature control is achieved. To demonstrate its utility, the temperature-controlled microfluidic flowcell is coupled to a prism-based TIRF microscope and is used to investigate the temperature-dependence of ribosome and transfer RNA (tRNA) structural dynamics that are required for the rapid and precise translocation of tRNAs through the ribosome during protein synthesis. Our studies reveal that the previously characterized, thermally activated transitions between two global conformational states of the pre-translocation (PRE) ribosomal complex persist at physiological temperature. In addition, the temperature-dependence of the rates of transition between these two global conformational states of the PRE complex reveal well-defined, measurable, and disproportionate effects, providing a robust experimental framework for investigating the thermodynamic activation parameters that underlie transitions across these barriers.

Interrupted Catalysis: The EF4 (LepA) Effect on Back-Translocation

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST→I1→I2→I3→PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3′-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I3 rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis.

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic.

Kinetically Competent Intermediates in the Translocation Step of Protein Synthesis

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.

Reverse Translocation of tRNA in the Ribosome

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.