Fluctuations of transfer RNAs between classical and hybrid states

Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg(2+)], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.     

Single-molecule study of viomycin's inhibition mechanism on ribosome translocation

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.     

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.     

Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations

Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A‐site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pretranslocation subpopulations are competent in real‐time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways. Proteins 2014; 82:1–9. © 2013 Wiley Periodicals, Inc.     

Binding of the peptide deformylase on the ribosome surface modulates the exit tunnel interior

Proteosynthesis on ribosomes is regulated at many levels. Conformational changes of the ribosome, possibly induced by external factors, may transfer over large distances and contribute to the regulation. The molecular principles of this long-distance allostery within the ribosome remain poorly understood. Here, we use structural analysis and atomistic molecular dynamics simulations to investigate peptide deformylase (PDF), an enzyme that binds to the ribosome surface near the ribosomal protein uL22 during translation and chemically modifies the emerging nascent peptide. Our simulations of the entire ribosome-PDF complex reveal that the PDF undergoes a swaying motion on the ribosome surface at the submicrosecond timescale. We show that the PDF affects the conformational dynamics of parts of the ribosome over distances of more than 5 nm. Using a supervised-learning algorithm, we demonstrate that the exit tunnel is influenced by the presence or absence of PDF. Our findings suggest a possible effect of the PDF on the nascent peptide translocation through the ribosome exit tunnel.     

An in vitro analysis of the dominance of emetine sensitivity in Chinese hamster ovary cell hybrids

The behavior of ribosomes derived from EmtR X EmtS hybrid cells in in vitro protein synthesis is similar to that observed with a 1:1 mixture of ribosomes from EmtR and EmtS cells. When mRNA (BM virus RNA) is present in limiting amounts (RNA/ribosome molar ratio = 0.1), protein synthesis in either mixture is sensitive to emetine. In contrast, when mRNA is present in excess (RNA/ribosome molar ratio = 2), the emetine resistant as well as the sensitive components are both expressed in the mixtures. These results strongly indicate that emetine resistant and sensitive ribosomes are present in the hybrid cells in about equal amounts and that the dominance of emetine sensitivity is best explained by assuming that emetine acts by blocking ribosome movement along mRNA by inhibiting the translocation step. The observed time lag in the expression of EmtRI and EmtRII mutations following mutagenesis is consistent with the above hypothesis for the mechanism of action of emetine.     

Applications of the molecular dynamics flexible fitting method

In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex.     

Single-Molecule Analysis of Translational Dynamics

Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all phases of the translation cycle. However, the relationship between structural dynamics, composition, and function remains unknown. The multistep nature of each stage of the translation cycle results in rapid desynchronization of individual ribosomes, thus hindering elucidation of the underlying mechanisms by conventional bulk biophysical and biochemical methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome and their impact on translational control. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.Translation and its regulation are intrinsically dynamic processes. In all organisms, to initiate translation, ribosomes must assemble from isolated subunits and an initiator transfer RNA (tRNA) on a messenger RNA (mRNA) at a specific start codon to establish a reading frame; protein factors guide this process. Elongation occurs through selection by the ribosome of cognate aminoacyl tRNAs, subsequent positioning of tRNAs for peptide bond formation chemistry, and movements of the tRNAs and mRNAs with respect to the codon (translocation). The directional process is iterative until termination at a stop codon, where the protein chain is released, and the ribosomal particle disassembled and recycled. Multiple ribosomes form higher-order polysomes on a single mRNA, with their own intrinsic dynamics.Dynamics are central to the mechanism and control of translation. Here we explicitly define dynamics as time-dependent changes in either composition or conformation of the translational machinery. Conformational dynamics in chemical systems are governed by an array of processes with vastly different timescales. Generally, dynamic processes become slower as they involve larger numbers of atoms. These range from electronic motions (timescale 10⁻¹⁴ sec), bond vibrations (10⁻¹³–10⁻¹² sec), through protein side chain or nucleic acid base/sugar local conformational changes (10⁻¹¹–10⁻⁶ sec), to larger conformational rearrangements (domain movements, etc.; 10⁻⁶–10² sec) that are often functionally cooperative. Compositional dynamics are determined by bimolecular association and dissociation rate constants: bimolecular arrival rates for ligands are governed by intermolecular collision frequencies, electrostatic interactions, and proper binding orientations for productive binding events, whereas dissociation rates are governed by energy barriers for dissociation of noncovalent intermolecular interactions.Fluctuations in molecular conformation and composition must be harnessed by the ribosome for accurate and rapid translation. The timescales of these dynamic changes dictate the overall rates of translation initiation and elongation: 0.2–0.5 initiation events/sec and elongation rates of 20–40 amino acids/sec in vivo, 1–5 amino acids/sec in vitro (Dennis and Bremer 1974a,b; Underwood et al. 2005). The ribosome uses external sources of free energy during translation—ATP hydrolysis during eukaryotic scanning, GTP hydrolysis by initiation, elongation and termination factors, and peptide bond formation. The free energy released by these irreversible reactions is used to drive the fidelity of initiation and elongation and the directional movement of the ribosome during both processes. The ribosome is thus a molecular motor.The link between ribosome and ligand dynamics and the control of protein synthesis remains a key mystery of translation. The past decade has witnessed the three-dimensional structures of prokaryotic, archaeal, and eukaryotic ribosomes at atomic resolution. How factors, tRNA, and ligands interact with the ribosome has been revealed by cryo-electron microscopy (cryo-EM) (at lower 6–12 Å resolution) and X-ray diffraction studies (as low as 2.5 Å for 30S from Thermus thermophilus [Kurata et al. 2008] and 2.4 Å for 50S from Haloarcula marismortui [Ban et al. 2000]). These structures have shown how GTPase factors engage with the 70S ribosomes at a conserved factor-binding site on the large subunit to mediate GTPase activity and subsequent conformational changes. Another key observation of early cryo-EM and more recent structural studies is that the ribosome adopts two general intersubunit conformations, related by a 6° rotation of the two subunits (Valle et al. 2003; Schuwirth et al. 2005; Agirrezabala et al. 2008; Zhang et al. 2009; Fischer et al. 2010; Dunkle et al. 2011). Peptide bond formation leads to a counterclockwise rotation of the small subunit with respect to the large subunit, and EF-G in the GTP form binds to this state. The peripheral domain L1 region of the ribosome was observed structurally to change its state in correlation with the two ribosomal conformations (Valle et al. 2003; Schuwirth et al. 2005; Agirrezabala et al. 2008), suggesting a coupling of domains within the ribosome. The intersubunit conformation of the ribosome was also correlated by EM to the relative orientations of tRNAs: In the nonrotated state (locked conformation), tRNAs are observed in the classical P site and A site, whereas in the rotated state (unlocked conformation) the tRNAs are in the Noller hybrid states with the 3′ ends of the tRNAs moved to the E and P sites and their respective anticodons in the P and A sites (Agirrezabala et al. 2008). These static structural views suggested a further correlation of tRNA and ribosome conformation during translation.The structural snapshots and prior biochemical studies are suggestive of dynamics during translation, yet experimental methods with resolution in real time are required to observe them directly. Here we focus on single-molecule methodologies that have provided an unprecedented view into the dynamics of prokaryotic translation. In the future, the same techniques can be applied to the study of eukaryotic translation dynamics.     

Motion of transfer RNA from the A/T state into the A‐site using docking and simulations

The ribosome catalyzes peptidyl transfer reactions at the growing nascent polypeptide chain. Here, we present a structural mechanism for selecting cognate over near‐cognate A/T transfer RNA (tRNA). In part, the structural basis for the fidelity of translation relies on accommodation to filter cognate from near‐cognate tRNAs. To examine the assembly of tRNAs within the ribonucleic–riboprotein complex, we conducted a series of all‐atom molecular dynamics (MD) simulations of the entire solvated 70S Escherichia coli ribosome, along with its associated cofactors, proteins, and messenger RNA (mRNA). We measured the motion of the A/T state of tRNA between initial binding and full accommodation. The mechanism of rejection was investigated. Using novel in‐house algorithms, we determined trajectory pathways. Despite the large intersubunit cavity, the available space is limited by the presence of the tRNA, which is equally large. This article describes a “structural gate,” formed between helices 71 and 92 on the ribosomal large subunit, which restricts tRNA motion. The gate and the interacting protein, L14, of the 50S ribosome act as steric filters in two consecutive substeps during accommodation, each requiring: (1) sufficient energy contained in the hybrid tRNA kink and (2) sufficient energy in the Watson–Crick base pairing of the codon–anticodon. We show that these barriers act to filter out near‐cognate tRNA and promote proofreading of the codon–anticodon. Since proofreading is essential for understanding the fidelity of translation, our model for the dynamics of this process has substantial biomedical implications. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Crystal structure of the ribosome recycling factor bound to the ribosome

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.     

Timing of GTP binding and hydrolysis by translation termination factor RF3

Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.     

Modelling competition and hybridization between native cutthroat trout and nonnative rainbow and hybrid trout

Native salmonid fish have been displaced worldwide by nonnatives through hybridization, competition, and predation, but the dynamics of these factors are poorly understood. We apply stochastic Lotka–Volterra models to the displacement of cutthroat trout by rainbow/hybrid trout in the Snake River, Idaho, USA. Cutthroat trout are susceptible to hybridization in the river but are reproductively isolated in tributaries via removal of migratory rainbow/hybrid spawners at weirs. Based on information-theoretic analysis, population data provide evidence that hybridization was the primary mechanism for cutthroat trout displacement in the first 17 years of the invasion. However, under some parameter values, the data provide evidence for a model in which interaction occurs among fish from both river and tributary subpopulations. This situation is likely to occur when tributary-spawned cutthroat trout out-migrate to the river as fry. The resulting competition with rainbow/hybrid trout can result in the extinction of cutthroat trout even when reproductive segregation is maintained.     

Modelling competition and hybridization between native cutthroat trout and nonnative rainbow and hybrid trout

Native salmonid fish have been displaced worldwide by nonnatives through hybridization, competition, and predation, but the dynamics of these factors are poorly understood. We apply stochastic Lotka-Volterra models to the displacement of cutthroat trout by rainbow/hybrid trout in the Snake River, Idaho, USA. Cutthroat trout are susceptible to hybridization in the river but are reproductively isolated in tributaries via removal of migratory rainbow/hybrid spawners at weirs. Based on information-theoretic analysis, population data provide evidence that hybridization was the primary mechanism for cutthroat trout displacement in the first 17 years of the invasion. However, under some parameter values, the data provide evidence for a model in which interaction occurs among fish from both river and tributary subpopulations. This situation is likely to occur when tributary-spawned cutthroat trout out-migrate to the river as fry. The resulting competition with rainbow/hybrid trout can result in the extinction of cutthroat trout even when reproductive segregation is maintained.     

Synchronous dynamics and rates of extinction in spatially structured populations

We explore extinction rates using a spatially arranged set of subpopulations obeying Ricker dynamics. The population system is subjected to dispersal of individuals among the subpopulations as well as to local and global disturbances. We observe a tight positive correlation between global extinction rate and the level of synchrony in dynamics among thesubpopulations. Global disturbances and to a lesser extent, migration, are capable of synchronizing the temporal dynamics of the subpopulations over a rather wide span of the population growth rate r. Local noise decreases synchrony, as does increasing distance among the subpopulations. Synchrony also levels off with increasing r: in the chaotic region, subpopulations almost invariably behave asynchronously. We conclude that it is asynchrony that reduces the probability of global extinctions, not chaos as such: chaos is a special case only. The relationship between global extinction rate, synchronous dynamics and population growth rate is robust to changes in dispersal rates and ranges.     

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.     

A fully coupled, mechanistic model for infectious disease dynamics in a metapopulation: movement and epidemic duration

The drive to understand the invasion, spread and fade out of infectious disease in structured populations has produced a variety of mathematical models for pathogen dynamics in metapopulations. Very rarely are these models fully coupled, by which we mean that the spread of an infection within a subpopulation affects the transmission between subpopulations and vice versa. It is also rare that these models are accessible to biologists, in the sense that all parameters have a clear biological meaning and the biological assumptions are explained. Here we present an accessible model that is fully coupled without being an individual-based model. We use the model to show that the duration of an epidemic has a highly non-linear relationship with the movement rate between subpopulations, with a peak in epidemic duration appearing at small movement rates and a global maximum at large movement rates. Intuitively, the first peak is due to asynchrony in the dynamics of infection between subpopulations; we confirm this intuition and also show the peak coincides with successful invasion of the infection into most subpopulations. The global maximum at relatively large movement rates occurs because then the infectious agent perceives the metapopulation as if it is a single well-mixed population wherein the effective population size is greater than the critical community size.     

Interplay of the Bacterial Ribosomal A-Site,S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome.