Ribosomal Motions Research Articles

Allosteric Effects of EF-G Domain I Mutations Inducing Ribosome Frameshifting Revealed by Multiplexed Force Spectroscopy.

Ribosome translocation catalyzed by elongation factor G (EF-G) is a critical step in protein synthesis where the ribosome typically moves along the mRNA by three nucleotides at each step. To investigate the mechanism of EF-G catalysis, it is essential to precisely resolve the ribosome motion at both ends of the mRNA, which, to our best knowledge, is only achieved with the magnetic-based force spectroscopy developed by our groups. Here, we introduce a novel multiplexed force spectroscopy technique that, for the first time, offers single-nucleotide resolution for multiple samples. This technique combines multiple acoustic force generators with the smallest atomic magnetometer designed for biological research. Utilizing this technique, we demonstrate that mutating EF-G at the GTP binding pocket results in the ribosome moving only two nucleotides on both ends of the mRNA, thereby compromising ribosome translocation. This finding suggests a direct link between GTP hydrolysis and ribosome translocation. Our results not only provide mechanistic insights into the role of GTP binding pocket but also illuminate how allosteric mutations can manipulate translocation. We anticipate broader applications of our technique in the ribosome field, leveraging its high efficiency and single-nucleotide resolution.

Divergent pathways of ribosomal motions during protein synthesis

Divergent pathways of ribosomal motions during protein synthesis

Theoretical Estimates of Physical Aspects Involved in the Power Stroke Hypothesis for Ribosome Translocation

The power stroke mechanism for ribosome translocation is evaluated through calculations of basic aspects of the mechanics, hydrodynamics, and elasticity involved in such a process. The results show that reported power stroke magnitudes would generate several physical problems for ribosome translocation: unwanted or disproportionate displacements of the ribosome and mRNA, destabilizing competing forces and high tension on the mRNA in the ribosome tunnel. All these issues can be related to the ribosome losing its correct reading frame during mRNA translation. To improve ribosome translocation models, the suggestion is to change the focus from tRNA–mRNA forced displacement to ribosome motion along the mRNA. Also, to consider that the mRNA is essentially different from cytoskeleton fibers and that the ribosome does not work alone during mRNA translation.

Structural defects of hypopseudouridylated ribosome observed during IRES-mediated translation initiation.

Pseudouridine is an abundant chemical modification found on major functional RNA molecules. Loss of pseudouridine is linked to diseases such as the X-linked dyskeratosis congenita (X-DC). While there is rich biophysical data on pseudouridine in model RNA molecules that show its stabilization role, little is known on its roles in intact functional RNA such as those within ribosome. To fill this gap, we studied structures of yeast ribosome isolated from cells that lack ribosome pseudouridine synthesis capability. We performed cryoEM structural analysis of hypopseudouridylated ribosome during initiation by the Taura syndrome virus (TSV) IRES, as the IRES-mediated translation has been found to be severely impaired in X-DC patient cells or by ribosome isolated from them. We found that though the overall fold of hypopseudouridylated ribosome remains similar to native ribosome, high resolution features reveal significant loss of pseudouridine-mediated interactions through water and long-range base pairings. Strikingly, when bound with TSV IRES and in presence of the elongation factor, eEF2, and GTP hydrolysis, the hypopseudouridylated ribosome favors an unconducive conformation for decoding linked to the loss of heavily pseudouridylated helix H69 and a hypermodified P site uridine, consistent with its reduced rate and fidelity of translation. Our data suggest that loss of pseudouridine in key ribosome sites alters the evolutionarily tuned ribosome motions required for IRES-mediated translation.

Stochastic microswimming model of ribosome motion on the polysome.

In this work we assume that the ribosome propels itself during the translocation step of the translation process of protein synthesis by running a cycle of stochastically generated conformational changes involving its two subunits. This cycle includes only two experimentally found ribosome shape changes. The main result is an analytic expression for ribosome's average swimming speed on a polysome, where the ribosome is in the presence of other ribosomes. Relevant geometric parameters of ribosome deformations are calculated first by solving a deterministic problem where the ribosome runs a cycle of prescribed conformational changes. The method of reflections and pairwise additivity are used to obtain the stresses and forces needed to apply the multiparticle reciprocal theorem. Ribosome's average velocity when it runs the corresponding stochastic cycle of deformations is calculated assuming independence among the conformational cycles of different ribosomes on the polysome. The results obtained show that swimming in tandem on the polysome allows the ribosome to reach any typical subcellular speed with deformations whose amplitude is of a smaller size than when it swims alone in the fluid. Also, the flow organized by its swimming stroke becomes more determinant for its motion than random diffusion, compared to the solitary ribosome.

Dynamic Behavior of Biomaterials Uncovered by Cryo-electron Microscopy.

Structural biology develops rapidly with time. The static structure analysis of biomaterials is not enough to satisfy the studies of their functional mechanisms, with a huge obstacle for the dynamic process of biological complexes. The rapid development of cryo-electron microscopy (cryo-EM) technology makes it possible to observe the near-atomic resolution structures and dynamic nature of biological macromolecules, in the fields of dynamic characteristics of proteins, protein-protein interactions, molecular recognition, and structure-based design. In this review, we systematically elaborate the contribution of cryo-EM technology in the field of biomaterials such as ribosome motion, membrane protein structure and conformational space, dynamic transmission within the plasma membrane and clinical applications. We also put forward a new standpoint in the development of cryo-EM technology.

An Efficient Timer and Sizer of Biomacromolecular Motions

Life ticks as fast as how proteins move. Computationally expensive molecular dynamics simulation has been the only theoretical tool to gauge the time and sizes of these motions, though barely to their slowest ends. Here, we convert a computationally cheap elastic network model (ENM) into a molecular timer and sizer to gauge the slowest functional motions of structured biomolecules. Quasi-harmonic analysis, fluctuation profile matching, and the Wiener-Khintchine theorem are used to define the "time periods," t, for anharmonic principal components (PCs), which are validated by nuclear magnetic resonance (NMR) order parameters. The PCs with their respective "time periods" are mapped to the eigenvalues (λENM) of the corresponding ENM modes. Thus, the power laws t(ns)= 56.1λENM-1.6 and σ2(Å2)= 32.7λENM-3.0 can be established allowing the characterization of the timescales of NMR-resolved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.

Ribosome Structure, Function, and Early Evolution.

Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S–50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

Quantitative Characterization of Domain Motions in Molecular Machines.

The work of molecular machines such as the ribosome is accompanied by conformational changes, often characterized by relative motions of their domains. The method we have developed seeks to quantify these motions in a general way, facilitating comparisons of results obtained by different researchers. Typically there are multiple snapshots of a structure in the form of pdb coordinates resulting from flexible fitting of low-resolution density maps, from X-ray crystallography, or from molecular dynamics simulation trajectories. Our objective is to characterize the motion of each domain as a coordinate transformation using moments of inertia tensor, a method we developed earlier. What has been missing until now are ancillary tools that make this task practical, general, and biologically informative. We have provided a comprehensive solution to this task with a set of tools implemented on the VMD platform. These tools address the need for reproducible segmentation of domains, and provide a generalized description of their motions using principal axes of inertia. Although this methodology has been specifically developed for studying ribosome motion, it is applicable to any molecular machine.

Theoretical estimate of the effect of thermal agitation on ribosome motion generated by stochastic microswimming

The effect of thermal agitation on ribosome motion is evaluated through the Péclet number, assuming that the ribosome is self-propelled along the mRNA during protein synthesis by a swimming stroke consisting of a cycle of stochastically-generated ribosome configurations involving its two subunits. The ribosome velocity probability distribution function is obtained, giving an approximately normal distribution. Its mean and variance together with an estimate of the in vivo free diffusion coefficient of the ribosome and using only configuration changes of small size, give a Péclet number similar to motor proteins and microorganisms. These results suggest the feasibility of the stochastic microswimming hypothesis to explain ribosome motion.

Unraveling the Mystery of Ribosome Induced RNA Unfolding

During translation, the ribosome encounters various structures in the mRNA from simple hairpins to more complex tertiary RNA folds. These structures must be unwound for accurate decoding and translation to proceed. To probe the unwinding activity of the ribosome, we prepared dye labeled RNA transcripts and dye labeled ribosomes to directly report on the folded/unfolded states of the RNA at various stages along the translational elongation cycle. Consistent with previous studies, we found that the ribosome was sufficient to resolve RNA structures in the absence of any additional factors. Surprisingly, significant unwinding was observed in the absence of translocation or motion of the ribosome along the mRNA and can be attributed to dynamic motions within and between the two ribosomal subunits. This suggests that thermal energy resident within these ribosomal motions is sufficient to lower the energetic barrier of unfolding. The extent of unwinding was observed to depend on the relative stability of the RNA structure used and the number of tRNAs present in the ribosome. Differences between unfolding of these RNA structures in the presence and absence of the ribosome will be discussed.

Rapid and Stable Transfer RNA Translocation through the Ribosome Ensured by Specific Contact Mechanisms

We combined high-resolution crystal structures with cryo-EM maps of 13 intermediate states of ribosomal factorless spontaneous retro-translocation to obtain structures of the 70S ribosome in each of the 13 states in atomic detail. We then performed 100 ns all-atom, explicit-solvent molecular dynamics simulations for each of these states. Intrinsic rates for key ribosomal motions between the states were estimated from the short time fluctuations of the L1-stalk, the tRNAs and intersubunit rotations. The rates revealed rapid, sub-microsecond motions of the L1-stalk and the 30S subunit. Surprisingly, it is tRNA motions, rather than large-scale intersubunit rotations, that are rate limiting for most transitions. The interaction free energy profile of the L1-stalk with the tRNA obtained from additional umbrella sampling simulations of the L1-stalk/tRNA interactions revealed an active role of the L1-stalk in pulling the tRNA from the P to the E site. Further, by detailed analysis of the frequency of contacts between 50S ribosomal proteins L5 and L16 and the tRNAs, we identified specific residues which guide the tRNAs between the binding sites. A sequence analysis of the L1, L5 and L16 proteins revealed that the conservation score for contacting residues is significantly above average. Different types of contacts characterize the interplay of these proteins with the tRNAs and involve 1) sliding of L5 residues along the tRNA elbow 2) stepping of the tRNA between L16 contact patches 3) final pulling of the tRNA by the L1-stalk. These contact mechanisms can explain how both rapid translocation and a stable tRNA binding affinity can be achieved despite large-scale displacements.

Ribosomal Kinetics and Concerted Motions from Nanoseconds to Seconds

During the elongation cycle, after peptide-bonds are formed in the ribosome, transfer RNAs translocate to their new binding sites. Combining high-resolution crystal structures, cryo-EM data of multiple translocation intermediates of factorless retro-translocation, and atomistic molecular dynamics simulations, we have analysed collective motions, intrinsic time scales, and overall transition rates of ribosomal motions promoting tRNA translocation.From a Marcus-Theory type transition state analysis of fast molecular fluctuations, unexpectedly fast sub-microsecond intrinsic rates were resolved for body and head rotations as well as swiveling motions. The tRNA transitions between A,P, and E site are seen to be clearly slower than microseconds and seem to determine the overall transition rate between the intermediates. Remarkably, our kinetic analysis recovers the sequence of intermediates proposed in Fischer et al. that was purely based on structural similarity, thereby adding time information to these data.Together with the millisecond dynamics revealed from single molecule studies and the slow dynamics between pre and post states, a Frauenfelder-type hierarchy of time scales and corresponding free energy barriers emerges, underscoring the notion of the ribosome as a stochastic molecular machine.

Single-Molecule Study of Ribosome Hierarchic Dynamics at the Peptidyl Transferase Center

During protein biosynthesis the ribosome moves along mRNA in steps of precisely three nucleotides. The mechanism for this ribosome motion remains elusive. Using a classification algorithm to sort single-molecule fluorescence resonance energy transfer data into subpopulations, we found that the ribosome dynamics detected at the peptidyl transferase center are highly inhomogeneous. The pretranslocation complex has at least four subpopulations that sample two hybrid states, whereas the posttranslocation complex is mainly static. We observed transitions among the ribosome subpopulations under various conditions, including 1), in the presence of EF-G; 2), spontaneously; 3), in different buffers, and 4), bound to antibiotics. Therefore, these subpopulations represent biologically active ribosomes. One key observation indicates that the Hy2 hybrid state only exists in a fluctuating ribosome subpopulation, which prompts us to propose that ribosome dynamics are hierarchically arranged. This proposal may have important implications for the regulation of cellular translation rates.

Single-molecule Fluorescence Microscopy Study Of The Ribosome Translation Process

The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, observation and manipulation at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the study of translation kinetics of single ribosomes via the direct observation of fluorescent amino-acid incorporations.In order to study the kinetics of amino-acid incorporation inside the growing protein by the ribosome, we use a home-made total internal reflection single-molecule fluorescence microscope (TIRFM). The mRNA-ribosome complex is attached to a polyethylene glycol modified glass coverslip surface by a streptavidin-biotin linkage. The ribosome is labelled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL [1]. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. The protein synthesis is observed in real time as the labelled amino acids are incorporated into the polypeptidic chain by the co-localization of QD and Bodipy-FL fluorescence signals.We will discuss the future application of this technique to single-molecule observation of the translation process, proof reading or even protein folding.Reference[1] K. Perronet, P. Bouyer, N. Westbrook, N. Soler, D. Fourmy, and S. Yoshizawa. Journal of Luminescence, 127, 264, 2007.

Comparisons of experimental and computed protein anisotropic temperature factors

Because of its appealing simplicity, the anisotropic network model (ANM) has been widely accepted and applied to study many molecular motion problems: such as ribosome motions, the molecular mechanisms of GroEL-GroES function, allosteric changes in hemoglobin, motor-protein motions, and conformational changes in general. However, the validity of the ANM has not been closely examined. In this work, we use ANM to predict the anisotropic temperature factors of proteins obtained from X-ray and NMR data. The rich, directional anisotropic temperature factor data available for hundreds of proteins in the protein data bank are used as validation data to closely test the ANM model. The significance of this work is that it presents a timely, important evaluation of the model, shows the extent of its accuracy in reproducing experimental anisotropic temperature factors, and suggests ways to improve the model. An improved model will help us better understand the internal dynamics of proteins, which in turn can greatly expand the usefulness of the models, which has already been demonstrated in many applications.

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Interpreting Correlated Motions Using Normal Mode Analysis

With the increased popularity of normal mode analyses in structural biology, it is important to carefully consider how to best utilize the results for gaining biological insights without over interpretation. The discussion in this article argues that for the purpose of identifying correlated motions in biomolecules, a case separate from concomitant conformational changes of structural motifs, it is generally important to use a large number of normal modes. This is illustrated through three increasingly complex examples. The simplest case includes two bilinearly coupled harmonic oscillators and serves as a straightforward problem where the important considerations are explicit and transparent. The argument is then generalized to include a system of N-coupled harmonic oscillators and finally to a realistic biomolecule. Although a small number of normal modes are useful for probing structural flexibility, it is clear that a much larger number of modes are required for properly investigating correlated motions in biomolecules.

Hinge-Like Motions in RNA Kink-Turns: The Role of the Second A-Minor Motif and Nominally Unpaired Bases

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle.

Global ribosome motions revealed with elastic network model

The motions of large systems such as the ribosome are not fully accessible with conventional molecular simulations. A coarse-grained, less-than-atomic-detail model such as the anisotropic network model (ANM) is a convenient informative tool to study the cooperative motions of the ribosome. The motions of the small 30S subunit, the larger 50S subunit, and the entire 70S assembly of the two subunits have been analyzed using ANM. The lowest frequency collective modes predicted by ANM show that the 50S subunit and 30S subunit are strongly anti-correlated in the motion of the 70S assembly. A ratchet-like motion is observed that corresponds well to the experimentally reported ratchet motion. Other slow modes are also examined because of their potential links to the translocation steps in the ribosome. We identify several modes that may facilitate the E-tRNA exiting from the assembly. The A-site t-RNA and P-site t-RNA are found to be strongly coupled and positively correlated in these slow modes, suggesting that the translocations of these two t-RNAs occur simultaneously, while the motions of the E-site t-RNA are less correlated, and thus less likely to occur simultaneously. Overall the t-RNAs exhibit relatively large deformations. Animations of these slow modes of motion can be viewed at http://ribosome.bb.iastate.edu/70SnKmode.