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Proposed sequence of events during EF-Tu/GTP-promoted binding of aminoacyl-tRNA in terms of tRNA ligand positions and the ribosome locking-unlocking (closing-opening) concept. Aa, aminoacyl.
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High-speed atomic force microscopy (HS-AFM) is now materialized. It allows direct visualization of dynamic structural changes and dynamic processes of functioning biological molecules in physiological solutions, at high spatiotemporal resolution. Dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding...
This essay begins with a description of the founding years of Journal of General Physiology (JGP) and a historical overview of the content of the journal. It then turns to key conceptual articles published in JGP that advanced the field of membrane permeation and ion selectivity. Much of this information comes from reading the online archives of JG...
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... However, the necessity of alternation processes to explain ribosome translocation is underlined by the fact that other models use them. An example is the ribosome conceived as a conveying machine (Spirin 2009). ...
... As such, the power stroke mechanism belongs to the model of the ribosome as a conveying machine. In that model, the ribosome directionally passes the tRNA-mRNA complex through itself by exerting forces on it, drawing the mRNA chain from its 5'-to 3'-end (Spirin 2009). The Brownian ratchet mechanism proposes that the A-site tRNA diffuses to the P-site, where random motion is corrected by some chemical transition occurring in the post-translocation state. ...
... They do not assume that the ribosome pulls or pushes the tRNA or the mRNA. However, such is the proposal made by powerstroke models (Yin et al. 2019;Chen et al. 2016) and implied by the model of the ribosome as a conveying machine (Spirin 2009). Exerting a force to move the tRNA-mRNA complex would generate, in principle, problematic situations for ribosome dynamics, besides not explaining ribosome motion but only tRNA-mRNA translocation. ...
... However, the necessity of alternation processes to explain ribosome translocation is underlined by the fact that other models use them. An example is the ribosome conceived as a conveying machine (Spirin 2009). ...
... As such, the power stroke mechanism belongs to the model of the ribosome as a conveying machine. In that model, the ribosome directionally passes the tRNA-mRNA complex through itself by exerting forces on it, drawing the mRNA chain from its 5'-to 3'-end (Spirin 2009). The Brownian ratchet mechanism proposes that the A-site tRNA diffuses to the P-site, where random motion is corrected by some chemical transition occurring in the post-translocation state. ...
... They do not assume that the ribosome pulls or pushes the tRNA or the mRNA. However, such is the proposal made by powerstroke models (Yin et al. 2019;Chen et al. 2016) and implied by the model of the ribosome as a conveying machine (Spirin 2009). Exerting a force to move the tRNA-mRNA complex would generate, in principle, problematic situations for ribosome dynamics, besides not explaining ribosome motion but only tRNA-mRNA translocation. ...
A meaningful dilemma in ribosome translocation arising from experimental facts is that, although the ribosome-mRNA interaction force always has a significant magnitude, the ribosome still moves to the next codon on the mRNA. How does the ribosome move to the next codon in the sequence while holding the mRNA tightly? The hypothesis proposed here is that ribosome subunits alternate the grip of the ribosome on the mRNA, freeing the other subunit of such interaction for a while, thus allowing its motion to the following codon. Based on this assumption, a single-loop cycle of ribosome configurations involving the relative position of its subunits is elaborated. When its dynamic is modeled as a Markov network, it gives expressions for the average ribosome translocation speed and stall force as functions of the equilibrium constants among the proposed ribosome configurations. The calculations have a reasonable agreement with experimental results, and the succession of molecular events considered here is consistent with current biomolecular concepts of the ribosome translocation process. Thus, the alternative displacements hypothesis developed in the present work suggests a feasible explanation of ribosome translocation.
... The copyright holder for this preprint (which this version posted May 9, 2023. ; https://doi.org/10.1101/2023.05.09.540051 doi: bioRxiv preprint pair was interpreted as a key supporting evidence for the model suggesting that EF-G•GTP acts by converting thermally-driven conformational dynamics of the ribosome and tRNAs into ribosome translocation along mRNA [2,7,24]. ...
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.
... The same conditions that allow for liquid water also ensure the relative stability of biomolecules and, as we now understand, thermal (cf. Brownian) motion and ratcheting that plays a key role in driving the function of large biomolecules (Spirin, 2009), including those directly involved in the replication, translation, and transcription of information in DNA and RNA (i.e. polymerases, ribosomes). ...
Computation, if treated as a set of physical processes that act on information represented by states of matter, encompasses biological systems, digital systems, and other constructs, and may be a fundamental measure of living systems. The opportunity for biological computation, represented in the propagation and selection-driven evolution of information-carrying organic molecular structures, has been partially characterized in terms of planetary habitable zones based on primary conditions such as temperature and the presence of liquid water. A generalization of this concept to computational zones is proposed, with constraints set by three principal characteristics: capacity, energy, and instantiation (or substrate). Computational zones naturally combine traditional habitability factors, including those associated with biological function that incorporate the chemical milieu, constraints on nutrients and free energy, as well as element availability. Two example applications are presented by examining the fundamental thermodynamic work efficiency and Landauer limit of photon-driven biological computation on planetary surfaces and of generalized computation in stellar energy capture structures (a.k.a. Dyson structures). It is shown that computational zones involving nested structures or substellar objects could manifest unique observational signatures as cool far-infrared emitters. While this is an entirely hypothetical example, its simplicity offers a useful, complementary introduction to computational zones.
... It has been proposed that GTP hydrolysis induces a conformational change in EF-G that either actively moves the tRNAs through a "power stroke" (16)(17)(18)(19)(20)(21) or induces a structural rearrangement in the ribosome that facilitates tRNA movement (2,13,17,(22)(23)(24)(25)(26). Others have proposed that GTP hydrolysis is coupled to tRNA-mRNA movement in a Brownian ratchet mechanism of translocation, in which movement is driven by thermal energy and EF-G acts as a pawl to prevent backward movement (5,6,(27)(28)(29)(30)(31)(32)(33). Although EF-G accelerates translocation by at least 10,000-fold (16,34), GTP hydrolysis contributes only about a 50-fold rate enhancement (13,14,16,35). ...
... Some investigators have proposed that hydrolysis is coupled to a conformational change in EF-G that either actively moves the tRNAs and mRNA in a "power stroke" (17,19,20) or induces a conformational change in the ribosome that provides the energy for tRNA and/or mRNA movement (13,17,(22)(23)(24)(25)(26). Others have suggested a Brownian ratchet mechanism driven by thermal energy in which EF-G acts as a pawl to prevent backward movement of the tRNAs (5,6,(27)(28)(29)(30)(31)(32)(33). Still others have suggested mechanisms that combine elements of both (18,21,25,26,47). ...
Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3′-terminal pyrene–labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.
... The binding energies associated with peptidyl-tRNA recognition within the SSU P site upon complete translocation of mRNA, and the unidirectionality provided by deacyl-tRNA dissociation, represent the barrier imposed by the opposite side of the sawtooth that prevents backwards motion. The hyper-swivel model of translocation proposed provides a framework that is consistent with the ribosome as a Brownian ratchet (5,11,20,114,115). ...
Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism. We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.
... Such machines are suitable for testing the proposed theory at its current, initial stage. On the other hand, dynamic biological structures are molecular machines-the ribosome can be put as an example (Rodnina & Wintenmeyer, 2011;Spirin, 2002Spirin, , 2009. ...
Theoretical frames for analyzing information in biological and molecular multicomponent structures are proposed. The mathematical foundations of the proposal are presented. Both the information encoded in structures is defined and the method of calculating the amount of this information is introduced. The proposed approach is applied to the operation of a molecular multicomponent machine.
... This happened in 1954, a year after the publication of articles by J. Watson and F. Crick on the structure of DNA and its biological meaning [3,4], which laid the foundation for molecular biology. As Alexander Sergeevich wrote [5], he was at that time a graduate student at the A. N. Bach Institute of Biochemistry of the USSR Academy of Sciences, but actually worked in the new building of the Biological (then -Biology and Soil) Faculty of the M. V. Lomonosov Moscow State University on the Lenin Hills, which was officially opened on September 1, 1954 [6]. ...
... Since the noncoding ribosomal RNAs turned out to be the major RNA species of the cell, they attracted the greatest Spirin's attention [5]. However, he began with a study of the genomic RNA of the tobacco mosaic virus (TMV) because it could easily be isolated in large amounts*; in addition, its integrity could unambiguously be proven by the infectivity test [15]. ...
... Now the Aa-tRNA-binding site of the small subunit with a new codon installed in it is ready to accept the next Aa-tRNA. Thus, the ribosome works cyclically: each cycle begins with the binding of Aa-tRNA, continues with the synthesis of the next peptide bond, and ends with the translocation (a detailed description of these events, taking into account new data, can be found in the Spirin's textbook [26] and in his latest papers on this issue [2,5,27]). ...
Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.
... These can be thought of as dynamic Brownian machines (81), using ATP hydrolysis or other sources of energy to bias the direction and perform useful work. The concept of a thermal ratchet for rectifying Brownian motions is useful for many proteins that involve physical transport J o u r n a l P r e -p r o o f (109)(110)(111). This concept can be extended to include a broader range of protein functions by thinking in terms of kinetic asymmetry instead of spatial asymmety providing a mechanism by which chemical free-energy released by catalysis can drive molecular adaptation. ...
Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple 'movies' to be taken of proteins in action and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life's machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.
... In the early 1960s, he proposed a model of the translating ribosome, whereby the ribosome oscillates between locked and unlocked states during the elongation cycle of protein synthesis. He suggested that the principles of molecular Brownian ratchet machines could explain the unidirectional translocation of mRNA and bound transfer RNA (tRNA) in the ribosome (3). Structural mobility of the ribosome during translation was experimentally confirmed by Alex in collaboration with Lydia Gavrilova in 1976, by showing that thermal energy alone is sufficient to drive translation under special conditions in the absence of elongation factors and GTP (factor-free or "nonenzymatic" translation) (4). ...
