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The Synthesis of proteins upon ribosomes
... In the course of the next composite step (translocation), the following events are presumed to occur: (i) the discharged tRNA (in site P) is released from the ribosome, (ii) the fMet-AA-tRNA is shifted from site A to site P, and (iii) the ribosome moves the length of one codon along the mRNA in the 5'-to-3' direction (401). Translocation is catalyzed by an elongation factor (S2). ...
... After peptide bond formation, the newly formed peptidyl-tRNA (fMet-AA-tRNA in the first cycle of chain elongation) is located in site A (100,101,142; see also 218,388,401), and the discharged tRNA, which remains bound to the ribosome (225), probably in site P (complex V in Fig. 3). Translocation (represented schematically by the conversion of complex V to complex VI in Fig. 3) is a composite step catalyzed by the S2 factor. ...
... These findings indicate that S2 is not needed for dipeptidyl-tRNA formation but that it is required for tripeptidyl-tRNA formation. The data are in accord with the view that the action of S2 results in (i) release of the discharged tRNA, (ii) translocation of the peptidyl-tRNA from the A site to the P site, and (iii) movement of the ribosome along the mRNA the length of one codon (218,401). There is no cause to assume that translation LENGYEL AND SOLL of codons beyond the third would require additional factors. ...
... Although it has been superseded recently by a more detailed model, the Watson two-site model (276) goes a long way toward displaying the problems that must be solved by a translating ribosome. One involves the proper matching of tRNA to codon, and the other involves the coordinate movements of tRNA and mRNA through separate ribosomal Aand P-sites associated in this model with the acceptor (A) and donor (peptidyl [P]) functions of peptide bond formation. ...
... Two important refinements have been added to the Watson model (276) in recent years. One is the identification of a third functional site for tRNA binding, the so-called exit site (E-site). ...
... The second refinement is more complex because it concerns the multiplicity of ways in which the 30S and 50S ribosomal subunits bind tRNA during the intermediate states of the elongation cycle. The two ribosomal binding sites of Watson's model (276) were defined originally by the two states of susceptibility of peptidyl-tRNA to the action of puromycin. In contrast, attempts to visualize an elongation cycle that makes use of the subunit structure of ribosomes tend to generate a higher order of binding states for tRNA during the elongation cycle. ...
... The ribosome is an RNA-protein molecular machine that coordinates the vital process of protein synthesis in all living organisms 6 . ...
The spirochete bacterial pathogen Borrelia (Borreliella) burgdorferi (Bbu) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. Here we present the structure of the Bbu 70S ribosome obtained by single particle cryo-electron microscopy at 2.9 Å resolution, revealing a bound hibernation promotion factor protein and two genetically non-annotated ribosomal proteins bS22 and bL38. The ribosomal protein uL30 in Bbu has an N-terminal α-helical extension, partly resembling the mycobacterial bL37 protein, suggesting evolution of bL37 and a shorter uL30 from a longer uL30 protein. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energy predictions for antibiotics reflect subtle distinctions in antibiotic-binding sites in the Bbu ribosome. Discovery of these features in the Bbu ribosome may enable better ribosome-targeted antibiotic design for Lyme disease treatment.
... A number of factors exhibit GTPbinding properties and can thereby be considered as molecular switches. Translation follows the basic model sketched many years ago by Watson (Watson 1964) and consists of the following phases: initiation, elongation, termination and recycling. ...
Protein biosynthesis represents a dynamic process that takes place on the ribosome and is driven by translation factors. Some of these factors are GTP binding proteins. They possess a limited inherent GTPase activity that is stimulated by interactions with the ribosome in a region located on the large ribosomal subunit (GTPase associated region). This site comprises several 23S rRNA elements (L10/L11 rRNA binding region and sarcin-ricin loop) and r-proteins, such as L6, L11, L14, and the L7/L12 stalk. The latter corresponds to an extended feature of the 50S ribosomal subunit, encompassing multiple copies of protein L12 that are linked to the ribosomal RNA via L10. Numerous lines of evidence indicated that L12 is essential for both translation factor binding and stimulation of their GTPase activities. Functionally, L12 can be divided into an N-terminal domain (NTD) responsible for dimerization and interaction with L10, a C-terminal domain (CTD) necessary for factor-related functions, and an intervening flexible hinge.Crystallographic studies of 50S subunits and 70S ribosomes hitherto failed to disclose the structure of the L7/L12 stalk, most probably due to the high mobility of the L12 hinge region. Thus, a complex anticipated to exhibit less flexibility was designed. It encompassed L10 and the NTD of L12 from the hyperthermophilic bacterium Thermotoga maritima. In the three crystal structures obtained, L10 displayed a globular NTD connected by a flexible loop to a long C-terminal α-helix. The latter displayed different orientations relative to the L10 NTD in different crystal forms and harbored three consecutive binding sites for the L12 NTD dimers. The L12 NTDs formed dimers that fitted to a mode of dimerization reported for the protein in isolation, both in solution (Bocharov et al. 2004; Moens et al. 2005) and in crystalline environment (Wahl et al. 2000). In the crystal structure of isolated T. maritima L12, the hinge region of one protomer exhibited an α-helical shape, folded onto the L12 NTDs of the dimer, while in tmaL10:(L12 NTD)6, the hinge was found replaced by the C-terminal α-helix of L10. Thus, it is likely that in complex with L10, the L12 hinges are flexible and unstructured, in agreement with several studies of this protein in solution.The in situ structure of an archaeal L10 NTD (a collaborative work with F. Schlünzen, J.M. Harms, Hamburg), enabled the positioning of the isolated tmaL10:(L12 NTD)6 complex on the 50S ribosomal subunit. The resulting model of a 50S subunit bearing a L10:(L12 NTD)6 complex was confirmed by an excellent fitting into the cryo-EM envelop of an E. coli 70S:EF-G:GDP:fusidic acid complex (N. Fischer, H. Stark, Göttingen). Based on these data and on structures of isolated L12, it was envisioned that the stalk is organized into three structural and functional elements, that are connected by flexible regions: (i) the stalk base, formed by the L10/L11 rRNA binding region, L11 and the L10 NTD, serving as attachment site for peripheral components; (ii) the C-terminal α-helix of L10 in complex with L12 NTD dimers that constitute a movable platform carrying L12 hinges and CTDs; (iii) the highly mobile L12 CTDs attached to the mobile platform via the hinge regions. This arrangement was in agreement with L12 CTDs being active players in the dynamic functions of the stalk.
... En 1964, James Watson, propose que l'ARN ribosomal établît des contacts directs avec les ARN de transferts et les ARN messagers. Le ribosome formerait le lieu de la traduction (Watson, 1964). En 1966, cette hypothèse est validée par Peter Moore (Moore et Asano, 1966). ...
La thréonylcarbamoylation de la base A37 des ARNt de type ANN (t⁶A) est universelle au sein des trois domaines du vivant car essentielle au bon fonctionnement de la cellule. La synthèse se catalyse en deux étapes, dont la première est assurée par la famille d’enzymes YrdC/Sua5, qui forme l'intermédiaire thréonyl-carbamoyle-AMP et la seconde par Kae1/OSGEP/Qri7/TsaD qui transfère le groupement thréonyl-carbamoyle du TC-AMP sur les ARNt substrats. La seconde famille d'enzymes requiert des partenaires protéiques différents selon le domaine du vivant. Le but de cette thèse était de comprendre les différents systèmes de biosynthèse du t⁶A au niveau moléculaire en utilisant des techniques biochimiques et structurales (la diffraction des rayons X (cristallographie), la diffusion des rayons X en solution (SAXS) et la cryo-microscopie électronique). La structure quaternaire du complexe TsaBDE de Thermotoga maritima a été résolue mettant en lumière les changements conformationnels de la sous-unité catalytique TsaD qui se traduit par une ouverture et une perturbation partielle du site actif induits par la présence de TsaE. Combinés à des données biochimiques, il apparaît que ce remodelage est nécessaire au recyclage du site catalytique. Chez l'Homme, la structure du sous-complexe OSGEP-LAGE3-GON7 a été résolue mettant en évidence que GON7, intrinsèquement désordonnée en solution, se structure au contact de LAGE3. L'étude structurale du complexe humain par SAXS combinée aux études génétiques et biochimiques montrent que GON7 stabilise le complexe sous forme pentamérique. Ces données ont été indispensable pour mieux comprendre les bases moléculaires d’une maladie génétique rare (le syndrome de Galloway-Mowat) causée par des mutations dans toutes les protéines de la voie de synthèse du t⁶A. Enfin, une cinquième sous-unité a été identifiée comme potentielle homologue de GON7 chez les Archées. Son étude structurale montre que la structure est différente de celle de GON7. Cependant, cette protéine stabilise le complexe sous forme pentamérique, mimant l'effet de GON7 chez les eucaryotes.
... We do not even know which of the component macromolecules (or parts thereof) are the more important. Unfortunately, we often think we understand translation, for we can speak of tRNA "adapters" (15) that are "translocated" from an "A site" to a "P site" (92). This view of translation (i) is too imprecise to provide a genuine molecular understanding of the process; (ii) because it is strong dogma, inhibits other attempts at such an understanding; and (iii) is probably misleadingly wrong. ...
... The functional role of the ribosome in protein synthesis was recently reviewed by Watson (223). A brief summary of some reactions which play a role in the elongation of the peptide chain is presented below. ...
... Along with the notion of messenger RNA had come the idea of the polyribosome-a single messenger RNA servicing a number of ribosomes at the same time. This notion of the polyribosome is represented in a familiar way in Fig. 3 (40,41). A messenger RNA molecule is depicted with a number of ribosomes attached to it. ...
... (Baldwin, 1953). Le ribosome possède trois sites distincts de liaison aux ARNt: les sites Aminoacyl (A) et Peptidyl (P) (Watson, 1964) et le site Exit (E) (Noll, 1966) (Figure 1). Le site A accueille l'ARNt aminoacylé entrant dans le ribosome après reconnaissance spécifique de son codon. ...
Structure, fonction et évolution de la famille universelle Sua5/YrdC impliquée dans la synthèse du nucléoside modifié t6ALa t6A est universellement présente au sein des ARNt décodant les codons ANN et est essentielle pour la fidélité de traduction. Sa synthèse se déroule en deux étapes, dont la première implique la formation de l’intermédiaire de réaction Thréonyl-Carbamoyl-AMP (TC-AMP) par la famille Sua5/YrdC. Cette famille est retrouvée chez tous les organismes et était donc vraisemblablement présente chez le dernier ancêtre commun universel (LUCA). Elle est composée de deux variants distincts, YrdC et Sua5, qui partagent un domaine catalytique orthologue. A la différence du variant YrdC qui est composé d’un domaine unique, le variant Sua5 possède un domaine C-terminal additionnel nommé SUA5, de fonction inconnue. La plupart des espèces code pour un seul variant et les deux variants sont présents dans les trois domaines du vivant, Eucaryote, Archée et Bactérie. Afin d’identifier le rôle du domaine SUA5 et du linker inter-domaine, nous avons étudié la protéine Sua5 de l’archée Pyrococcus abyssi. Nos résultats montrent que ces deux régions sont importantes pour l’activité de Sua5. Le linker est capable de contrôler le passage des ligands en changeant de conformation tandis que le domaine SUA5 agit comme une plateforme d’ancrage pour le linker. Afin de comprendre l’histoire évolutive de la famille Sua5/YrdC, nous avons ensuite étudié la distribution des variants et nous avons utilisé des approches in silico et in vitro afin de déterminer les différences fonctionnelles entre YrdC et Sua5. L’ensemble de ces données nous permet de proposer que LUCA possédait une protéine Sua5 et qu’YrdC serait apparu suite à une perte de domaine dans certains lignées lors de l’évolution.
... The 3'-CCA end of tRNAs is loaded with the correct amino acid by specific enzymes, the aminoacyl-tRNA synthetases. This last step of gene expression is catalyzed by the ribosome, in which tRNAs are selected, and progress along the mRNA through tRNA sites, named A-, P-and E-sites (Ben-Shem et al., 2010;Rheinberger et al., 1981;Watson, 1964). ...
The ribosome is a biomolecular machine essential for the survival of any organism due to its central role in protein synthesis. The characterization of its interactions with its many partners is a crucial element in better understanding the mechanisms of translation and inhibition in eukaryotes and prokaryotes. Inhibition of translation is a strategy used by many ribosome-targeting antibiotics to fight bacterial infections. Understanding their mode of action has become a global priority in addressing the problem of bacterial resistance. In eukaryotes, another strategy is used by viruses to block and appropriate the host's translational machinery through non-coding RNA structures (IRES) capable of directly recruiting the ribosome. Although widely characterized, few thermodynamic and kinetic data are available for these two ribosome interaction systems. My project is intended to use innovative biophysical approaches in order to provide an original view of the interactions of the E. coli ribosome with macrolides, and of the S. cerevisiae ribosome with the intergenic IRES of the CrPV.
... In many of the present biochemical textbooks the bacterial elongation cycle show only two different tRNA binding sites on the ribosome: the A site (aminoacyl or acceptor site) that binds the incoming ternary complex EF-T u·GTP·aa-tRNA, and the P site (peptidyl or donor site) with bound peptidyl-tRNA [Watson, 1964]. However, a third site, the E site (release or exit site [Wettstein and Noll, 1965]), that binds the deacylated tRNA before it leaves the ribosome is nowadays firmly established . ...
... On the other hand, there exist at least 20 different molecules of tRNA such that each one of them corresponds (or attaches) to a specific amino acid aggregate 11 . A complete 3D structure of tRNA was unknown 13 . However, the preliminary information about the structure of the tRNA mosaic was that it consists of a sequence of tiles (including A, U, G, C, or I) 11,14 . ...
In this paper, we continue investigating the structure of the Primary Language of the human brain introduced by J. von Neumann in 1957. According to our hypothesis, the Primary Language is the Language of Visual Streams (mental movies). Our investigation is focused on the major ancient algorithms essential for the development of humanity, which should have used the Primary Language directly, bypassing human symbolic languages. One of them is the Algorithm of Discovery (AD), the algorithm for inventing new algorithms. We investigate the AD by applying it to the past discoveries. In this paper, we apply it to rediscovering the “mechanics” of protein translation. We emphasize application of mosaic reasoning as one of the means for focusing visual streams in the desired direction. Specifically, we identify the matching rules that are used to link the mRNA and tRNA mosaics.
... A typical example is the fast transfer of digital layout data into physical structures during the writing of masks by using electron beams. An analog serial mechanism is also found in the synthesis of proteins [58][59][60][61][62]. The linear primary structure of natural peptides is the direct result of a serial fabrication process. ...
Besides the fundamental competition between the top-down and bottom-up approaches in nanotechnology, there are some basic aspects for organizing structures and functions at the molecular level. The recent challenges to the development of nanotechnology are marked by a group of general requirements: selection of suited building units, overcoming the restrictions of planar technology, shrinking of nanofabrication facilities, sustainable production and management of life cycles, organization of autonomy and communication at the nano-level, and the optimization of power consumption and energy management. Looking at the natural principles in the construction, synthesis, and function of proteins helps in understanding the principal differences between the currently applied technologies and the characteristics of biomolecular mechanisms in cells. This view allows formulating seven basic rules to meet the general requirements for future developments in molecular nanotechnology.
... The elongation step ( Fig. 1.1) is the most complicated and least understood aspect of protein synthesis. Even though the classical two-site model (Watson, 1964) used to describe the elongation step of protein synthesis has undergone some recent modifications (Moazed and Noller, 1989, reviewed in Ramakrishnan, 2002), the principle remains the same. Aminoacylated tRNA is brought into the A -site of the mRNAprogrammed ribosome as a ternary complex with elongation factor Tu (EF-Tu) and GTP. ...
... According to the classical definition the A site of the ribosome binds AcPhePhe-tRNAPhe in complex 111 [21]. The Phe-tRNAPhe of complex I1 is located at a site which has been given various names (see below), in particular, pre-A and A-like site [l -3, 211. ...
Ultraviolet(254 nm)-irradiation-induced cross-linkages in ribosomal complexes allowed identification of proteins in contact with tRNA at different elongation steps. Both the set and the ratio of cross-linked proteins, i.e. the structural characteristics of the tRNA-binding sites of the ribosome, were shown to depend strongly not only on the position of the mRNA codon with which tRNA interacts as a component of a ribosomal complex, but also on its functional state, i.e. on the elongation step. A new classification of tRNA-binding sites of ribosome is suggested.
This paper is partly a summary of the book ‘From a grain of salt to the ribosome’ [1 Olovsson I, Liljas A, Lidin S. From a grain of salt to the ribosome. Singapore: World Scientific; 2014; ISBN 978-9814623117.[Crossref] , [Google Scholar]], with extension on some points. Sometimes the developments in science may be very rapid and not fully appreciated at all corners of the scientific society. Harry Clary Jones was a well-known chemist at Johns Hopkins University at the turn of the previous century. He had, in his earlier days, spent time in the laboratories of Wilhelm Ostwald in Leipzig, Svante Arrhenius in Stockholm and Jacobus van’t Hoff in Amsterdam. He wrote many papers and twelve books. In 1913 he claimed in a book [2 Jones HC. A new era in chemistry – some of the more important developments in chemistry during the last quarter of a century. New York: D van Nostrand; 1913. [Google Scholar]]:
We do not know the formula of rock salt, or of ice; and we have no reliable means of finding out these simplest matters about solids. Our ignorance of solids is very nearly complete.
It is evident that he was unaware of the very recent developments and the revolution in chemistry that had just taken place with the birth of X-ray crystallography.
Ribosomes are remarkable ribonucleoprotein complexes that are responsible for protein synthesis in all forms of life. They polymerize polypeptide chains programmed by nucleotide sequences in messenger RNA in a mechanism mediated by transfer RNA. One of the most challenging problems in the ribosome field is to understand the mechanism of coupled translocation of mRNA and tRNA during the elongation phase of protein synthesis. In recent years, the results of structural, biophysical and biochemical studies have provided extensive evidence that translocation is based on the structural dynamics of the ribosome itself. Detailed structural analysis has shown that ribosome dynamics, like aminoacyl-tRNA selection and catalysis of peptide bond formation, is made possible by the properties of ribosomal RNA.
Ribosomes are intracellular ribonucleoprotein particles of about 200 Å in diameter which are essential components of the translation system in all organisms (Tissières and Watson, 1958; Tissières et al., 1959; Watson, 1964; Schlessinger and Apirion, 1969; Nomura, 1970). They are usually isolated from cell-free extracts by sedimentation at 105,000 × g for several hours and then purified from nonribosomal contaminants by high-salt washing (Kurland, 1971). They are characterized by their typical sedimentation properties and their ability to function in protein synthesis in vitro. Extensive physical studies of ribosomes have been carried out (Hill et al., 1969). All ribosomes which have been examined are made up of two subunits (small and large). The separation of the ribosome into subunits, which probably has physiological significance (the “ribosome cycle”), is accomplished by dialyzing bacterial ribosomes against low magnesium ion concentrations Staehelin and Maglott, 1971), or eukaryotic ribosomes against solutions containing concentrated potassium chloride (Martin et al., 1971; Staehelin and Falvey, 1971) or urea (Petermann, 1971). The subunits are then separated by sucrose density-gradient centrifugation (McConkey, 1967). The dissociation of ribosomes into subunits is reversible. The gross structure of a prokaryotic (bacterial) ribosome is shown in Figure 1. The structure of a typical eukaryote ribosome is very similar; however, ribosomes from prokaryotes and eukaryotes differ in size.
The elongation cycle of protein synthesis was formalized by Watson (1964), who proposed a simple two-site model that accounted for most of the experimental observations concerning the interactions between tRNA and ribosomes known at that time. The robustness of this classical model was apparent from the fact that it withstood nearly two decades of experimental testing. In the meanwhile, a number of studies led to proposals advocating additional ribosomal binding sites for tRNA. Eventually, it was recognized that a new feature, the E site, had to be accounted for (Rheinberger et al. 1981; Grajevskaja et al. 1982; Kirrilov et al. 1983; Lill et al. 1984), and so the two-site model was expanded to accomodate three tRNA binding sites, although the main features of the mechanism remained essentially unchanged (Fig. 1). Beginning with a peptidyl-tRNA (or initiator tRNA) in the P site (Fig. la), a new aminoacyl-tRNA is delivered to the A site via a tRNA EF-Tu-GTP ternary complex (Fig. lb). The roles of EF-Tu and GTP were recognized to be important for translational accuracy, and appear to involve a kinetic proofreading mechanism (Thompson 1988; Kurland et al. 1990). Once the A and P sites are filled, peptide bond formation occurs spontaneously, catalyzed by peptidyl transferase, an integral part of the large ribosomal subunit; in the classical model, this results in transfer of the nascent polypeptide chain to the A-site tRNA, leaving a deacylated tRNA in the P site (Fig. lc).
The process of translation (elongation) on the ribosome is composed of the repeating cycles, each consisting of three successive steps: aminoacyl-tRNA binding, transpeptidation, and translocation (Watson 1964; Lipmann 1969). The translocation step includes significant intraribosomal displacements of a template and the products of the transpeptidation reaction: the release of deacylated tRNA, the transport of peptidyl-tRNA from one site to the other, and the shift of the template polynucelotide by one codon.
At any given time in the course of polypeptide elongation, the ribosome is attached to the coding region of mRNA and retains the molecule of the peptidyl-tRNA (Fig. 9.1). The peptidyl-tRNA is a nascent peptide chain bound through its C-terminus to the tRNA that has donated the last amino acid residue to the peptide. Such a ribosome can bind or may become capable of binding the aminoacyl-tRNA determined by the next mRNA codon (Fig. 9.1 step I). The binding of the aminoacyl-tRNA results in the retained peptidyl-tRNA and the newly bound aminoacyl-tRNA being present on the ribosome simultaneously. Their side-by-side location and the catalytic activity of the ribosome are prerequisites of the transpeptidation reaction: the C-terminus of the peptidyl residue is transferred from the tRNA (to which it had previously been bound) to the amino group of the aminoacyl-tRNA (Fig. 9.1 step II). As a result, the formation of a new peptidyl-tRNA with the peptide elongated by one amino acid residue at the C-end takes place; the other product of the transpeptidation reaction is the deacylated tRNA. In order to make the ribosome competent to bind the next aminoacyl-tRNA, the intraribosomal ligands (tRNAs and mRNA) must be displaced, resulting in the vacation of a place for the aminoacyl-tRNA and in the positioning of the next mRNA codon (Fig. 9.1 step III); this step is called translocation.
In an actively growing culture of micro-organisms, ATP formed by the energy-yielding metabolism of the organisms is rapidly expended in a variety of metabolic processes. An appreciable amount of ATP is consumed in the biosynthesis of new cell components, including energy-storage compounds such as glycogen and poly-β-hydroxybutyrate; but there are other metabolic processes taking place in growing micro-organisms which also require ATP, and information on these additional items of ATP expenditure has gradually accumulated in recent years.
The ribosome is a cell organelle that is essential for protein synthesis. That the ribosomes provide sites for the binding of mRNA and the amino-acyl tRNA has been established (see reviews by Watson [1, 2]). In addition, the structural complexity of ribosomes suggests greater complexity in function and, in fact, there are several indications that the structure of ribosomes is profoundly involved in the translation of the genetic code [3, 4, 5]. Recent experiments also suggest that the peptidyl transferase is a part of 50 S ribosomal proteins [6, 7]. However, the detailed function of ribosomes in protein synthesis is still unclear. It appears that a comprehensive understanding of the mechanism of protein synthesis must await an elucidation of the structure and function of ribosomes.
In order to coordinate the complex biochemical and structural feat of converting triple-nucleotide codons into their corresponding amino acids, the ribosome must physically manipulate numerous macromolecules including the mRNA, tRNAs, and numerous translation factors. The ribosome choreographs binding, dissociation, physical movements, and structural rearrangements so that they synergistically harness the energy from biochemical processes, including numerous GTP hydrolysis steps and peptide bond formation. Due to the dynamic and complex nature of translation, the large cast of ligands involved, and the large number of possible configurations, tracking the global time evolution or dynamics of the ribosome complex in translation has proven to be challenging for bulk methods. Conventional single-molecule fluorescence experiments on the other hand require low concentrations of fluorescent ligands to reduce background noise. The significantly reduced bimolecular association rates under those conditions limit the number of steps that can be observed within the time window available to a fluorophore. The advent of zero-mode waveguide (ZMW) technology has allowed the study of translation at near-physiological concentrations of labeled ligands, moving single-molecule fluorescence microscopy beyond focused model systems into studying the global dynamics of translation in realistic setups. This chapter reviews the recent works using the ZMW technology to dissect the mechanism of translation initiation and elongation in prokaryotes, including complex processes such as translational stalling and frameshifting. Given the success of the technology, similarly complex biological processes could be studied in near-physiological conditions with the controllability of conventional in vitro experiments.
The crystal structure of tRNA, the topography of ribosomes, and the enzymology of peptide elongation are well known; however, although numerous models have been proposed to account for ribosome function, there is relatively little factual information on the details of the mechanism by which this is accomplished. Of primary importance for an understanding of this mechanism is to know the number, location, relative orientation, and time of movement of tRNA molecules in ribosomes during the reaction steps of peptide elongation. Early studies prompted Watson (1964) to propose the now classic two-site model to account for the basic functional requirements for peptide elongation, namely, to bring two tRNAs into reactive proximity to allow transfer of a nascent peptide from one tRNA to the free amino group of the incoming aminoacyl-tRNA and unidirectional, coordinated movement of tRNA and mRNA through a ribosome. This model, given in modified form to include the two GTP-dependent reactions promoted by the peptide elongation enzymes, EF-Tu and EF-G, is shown in Figure 28.1. The two tRNAs were presumed to be specifically bound to two sites defined originally in terms of peptidyl transferase reactions as the donor (peptidyl or P) site and acceptor (aminoacyl or A) site, respectively.
The ribosomal synthesis of a peptide bond takes place by transfer of the peptidyl ester of peptidyl-tRNA to the amino acid amino group of an incoming aminoacyl-tRNA. Attempts to isolate an acyl-ribosome intermediate of the kind found for many enzymecatalyzed hydrolyses or transpeptidation reactions have been unsuccessful. This has led many investigators to speculate that transpeptidation by the ribosome is brought about by an appropriate spatial orientation and alignment of the aminoacyl-tRNA and peptidyl-tRNA without the catalytic involvement of special nucleophilic groups of the large ribosomal subunit as discussed by Spirin (1986). The range of covalent derivatives other than peptides or amides that can be formed: esters (Fahnestock et al., 1970), thioesters (Gooch and Hawtrey, 1975), thioamides (Victorova et al., 1976), phosphinoamides (Tarussova et al., 1981) support this hypothesis with the implication that the peptidyl transferase reaction itself is of the SN2 type with nucleophilic substitutions through a tetrahedral intermediate. However, these considerations only serve to emphasize the importance of understanding how the 3’ ends of two tRNAs are brought precisely into reactive proximity to facilitate the reaction that by many measures is the most evolutionarily conserved and fundamental process of life, the reaction system by which genetic information encoded in nucleic acid is translated into protein.
Among the fundamental, trend-setting achievements in the quest for the understanding of the biosynthesis of proteins was the assertion that nucleic acids play a decisive rôle on this process. This hypothesis was advanced at the beginning of the 1940’s. Since then, the resolution of the biosynthesis of proteins has been inseparably linked with studies on the nucleic acids and their complexes with proteins.
This chapter presents albumin synthesis. Serum albumin is the major protein produced in the liver, comprising as much as 50% of the productive effort at any one moment. The concentration of this protein in the plasma has long been used as a bellwether of health and disease. It must be remembered that the serum albumin level is only the complex end result of synthesis, degradation, and distribution. Albumin is synthesized by the hepatocyte. It finds its way directly into the hepatic plasma and, hence, to the systemic circulation. Its half-time of survival is 20 days in man, and it is degraded in sites yet unknown. The most important factor regulating albumin synthesis is nutrition. Adequate nitrogen intake is basic to all the other mechanisms involved in regulation of protein synthesis. Thyroid hormone and cortisone stimulate albumin synthesis in vivo. When excess thyroid is administered to healthy patients, a sustained 30% increase in both albumin synthesis and albumin degradation occurs within 2 weeks.
Immunization of an animal with bacterial ribosomes generally elicits the formation of antibodies capable of precipitating not only the homologous ribosomes used for immunization but also ribosomes of different origin (Barbu et al., 1961; Panijel and Barbu, 1961). However, for a given antiserum, the qualitative aspects of the immune reaction vary with the species of bacteria being tested. In addition, as shown in Figure 1 (A, B, C), the amount of antibody precipitable by a given bacterial ribosome varies with the antiserum used. It is evident that ribosomes even from distant species possess common antigenic determinants, thus explaining the cross-reactions observed. With a given antiserum, such as the E. coli K12 ribosome antiserum, one can distinguish the following: (a) homologous ribosomes, e.g., those from various strains of E. coli or even other enteric bacteria; (b) close heterologous ribosomes, e.g., ribosomes of Proteus vulgaris; and (c) distant heterologous ribosomes, e.g., ribosomes of Clostridia. Such a “classification” of ribosomes relative to a given antiserum seems to be genetically significant. Indeed, McCarthy and Bolton (1963), who used the hybridization technique to study the relationship between messenger RNAs extracted from E. coli and DNAs of other origin, subsequently arrived at a classification in agreement with ours.
This chapter discusses the process of peptide chain elongation. For studies on the mechanism of polypeptide chain elongation extensive use has been made of the model system in which polyuridylic acid, poly(U), directs the formation of polyphenylalanyl-tRNA. The requirements for this in vitro system are high salt washed ribosomes, Phe-tRNA, poly (U), guanosine triphosphate, Mg2+, and NH4+ ions, a sulfhydryl reagent such as dithiothreitol and three protein factors which are present in the supernatant fraction of lysed cells. In peptide chain elongation, Protein growth is accomplished by a cyclic process involving aminoacyl-tRNA binding, peptidyl transfer, peptidyl tRNA translocation, and exposure of a new triplet codon through movement of the ribosome on mRNA. These factors have been recently designated as elongation factor thermo unstable (EF-Tu), elongation factor thermo stable (EF-Ts), and elongation factor G (EF-G), the new symbols being intended to replace the various designations used for the factors in different laboratories. When isolated from the soluble fraction of the cell, EF-Tu and EF-Ts are associated, and this complex is referred to as EF-T. The respective factors from various bacterial species may be interchanged in the partial reactions of peptide chain elongation as well as in the overall polymerization reaction. The amount of EF-T and EF-G in the bacterial cell is a significant percentage of the total soluble proteins. The EF-G content of Escherichia coli cells has been estimated under different growth conditions to be 2-3 % and 6% and the EF-T content as 2% and 3%. The relative amount of each factor compared to ribosomes remains constant at different growth rates, suggesting that the synthesis of the polypeptide chain elongation factors is coordinated with that of ribosomes.
The attachment of deacylated tRNA and [14C]Phe-tRNA to ribosomes in response to poly U was studied. The extent of binding of tRNAPhe to ribosomes is approximately equal to that of [14C]Phe-tRNA. Transfer RNA dilutes the binding of [14C]Phe-tRNA as predicted from the ratio of tRNA to aminoacyl-tRNA, suggesting that tRNAPhe and [14C]Phe-tRNA have approximately equal affinities for ribosomal binding sites. Under the conditions studied, tRNA and [14C]Phe-tRNA are released from poly U-ribosome complexes at relatively slow rates. The synthesis of phenylalanyl-puromycin peptides was detected at 0.02, but not 0.01 m-Mg2+. At 0.01 m-Mg2+, tRNA inhibits AUG-dependent binding of N-formyl-[3H]Met-tRNA to ribosomes and the formation of N-formyl-[3H]methionyl uromycin. These data show that the rate of codon recognition of initiator and other codons by aminoacyl-tRNA at two or more ribosomal sites may be affected by the ratio of tRNA to aminoacyl-tRNA.
50 and 30 s ribosomal particles were dissociated into smaller subribosomal particles (40 and 23 s “cores”) and “split proteins” by centrifugation in cesium chloride. The resultant core particles are inactive in protein synthesis. Mixing the split proteins with these core particles results in reconstitution of ribosomes which are active in in vitro polypeptide synthesis.
How We Got to Where We Are: the Ribosome in the 21st Century, Page 1 of 2
Abstract
This article is a short, informal history of the ribosome field that begins with the emergence of the field in the 1930s and ends with a description of its state in 2007, the year this essay was written. The growth in our understanding of both the role of the ribosome in protein synthesis and its structure is emphasized. Starting in 2000, the field experienced a massive upheaval as a result of the publication of the first atomic-resolution crystal structures for ribosomes. However, by 2007, the field had recovered sufficiently so that one could begin to understand how it was likely to evolve in its "poststructural" era. For that reason, this essay is about as useful as a short history of the ribosome field today as it was several years ago, when it was written.
On the basis of published data, a detailed model of the active centre of Escherichia coli peptidyl transferase is proposed. The major conclusions are as follows: A binding site is present at each of the acceptor (A′) and donor (P′) substrate binding sites of the enzyme for the 3′-terminal CpCpA of aminoacyl- and peptidyl-tRNA, respectively. In particular, the acceptor CpCpA binding site is composed of sites for the following groups: the terminal adenine, the first phosphoryl residue from the 3′-terminus, the 3′-penultimate cytosine, and the second 3′-CMP residue. In addition, two binding sites are present on each of the A′ and P′ sites, one for the basic and one for the hydrophobic aminoacyl R groups of both aminoacyl-tRNA and the carboxyl-terminal amino acid of peptidyl-tRNA. The role of these sites in the binding of inhibitors and substrates and in the mechanism of catalysis of peptide bond formation by peptidyl transferase is discussed.
Nascent proteins are extended into a tunnel or cavity within the large ribosomal subunit as they are formed by the successive addition of amino acids to their N-terminus. This process appears to be associated with the acquisition of secondary and tertiary structure that is important for folding into the native conformation or transport of the newly formed protein into membranes or other subcellular structures. This chapter discusses the aspects of the structure and function of ribosomes that contribute to these processes. The synthesis of proteins in all living cells is carried out by ribosomes that are composed of two structurally different subunits that associate upon initiation of protein biosynthesis. Ribosomes are massive entities comparable in size to large multienzyme complexes. They have unique structures composed of RNA and proteins of specific primary sequence. The nascent peptide provides the basis for the intimate relationship between active ribosomes and membranes in bacteria.
Die in der Basensequenz der Nucleinsäuren enthaltene Information für die Proteinstruktur wird am Ribosom in die Aminosäurensequenz der Proteine übersetzt. Diese Übersetzung, Translation genannt, läßt sich in den Kettenstart, die Kettenverlängerung und den Kettenabschluß gliedern. An jedem Abschnitt sind mehrere spezifische Proteinfaktoren und Nucleinsäuren beteiligt. – Beim Kettenstart werden aus der Startaminosäure-tRNA, der mRNA mit dem Startsignal und der kleinen und großen Untereinheit eines Ribosoms Startkomplexe gebildet. Dabei wirken GTP und die Startfaktoren mit. – Bei der Kettenverlängerung wird in einem Reaktionszyklus jeweils eine Aminosäure aus der Bindung an die tRNA in eine Bindung in der Polypeptidkette überführt. Zunächst wird die zu inkorporierende Aminosäure als Aminoacyl-tRNA an das Ribosom gebunden, wozu GTP und Proteinfaktoren erforderlich sind. Die anschließende Entstehung der Peptidbindung wird durch die Peptidyltransferase der großen Ribosomenuntereinheit katalysiert. Danach wird die nunmehr um eine Aminosäure verlängerte Peptidyl-tRNA auf dem Ribosom von der Aminosäure-Acceptorstelle A an die Peptidyl-Donorstelle P verlagert. Hierzu sind ein weiterer Proteinfaktor und die Spaltung von GTP in GDP und Phosphat notwendig. – Der Kettenabschluß wird eingeleitet, sobald eines der drei Terminatortripletts UAA, UAG oder UGA auf der sich relativ zum Ribosom vom 5′- zum 3′-Ende bewegenden mRNA das Ribosom erreicht. Die Ablösung der fertigen Polypeptidketten vom Ribosom hängt von den Ablösefaktoren ab. Vor dem Start einer neuen Polypeptidkette dissoziieren die Ribosomen in ihre Untereinheiten.
Polypeptide chain elongation is conveniently described in three separate steps. Much is now known about the functioning and structures of molecular components involved in each step but little is known about the kinetics of the process and its motive force. This is the second article in our three-part series on protein synthesis.
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Action of heated RNAs from reticulocytes and liver nuclei on the cell-free synthesis of hemoglobin. RNAs from rabbit reticulocytes and liver nuclei and from guinea-pig liver nuclei, as well as the fractions obtained from these RNAs by centrifugation in a sucrose gradient, have been heated at 90° for 30 min and added to cell-free systems from reticulocytes in order to observe their action on hemoglobin biosynthesis. 1. 1. Heating increases the stimulating effect of reticulocyte RNA on hemoglobin biosynthesis; it very slightly increases the stimulating effect of rabbit liver RNA and has no effect on the RNA from guinea-pig liver. 2. 2. Heating of ribosomal RNAs from reticulocytes suppresses their inhibiting effect on hemoglobin biosynthesis; it has little effect on ribosomal RNA from rabbit liver and no effect on ribosomal RNA from guinea-pig liver. 3. 3. Heating of RNAs from rabbit reticulocytes and liver which sediment between 16 S and 4 S, and which probably contain messenger RNA for hemoglobin, does not modify their stimulating effects on hemoglobin biosynthesis. 4. 4. If messenger RNA is removed from reticulocyte ribosomes by elimination of magnesium, the 28-S RNA prepared from these ribosomes is not modified by heating. If this 28-S RNA is incubated with a preparation rich in messenger RNA and reisolated, it becomes an activator of hemoglobin synthesis by heating. These observations can be explained by the existence of specific bonds between the messenger RNA of rabbit hemoglobin and ribosomal RNA from rabbit reticulocytes. These is little affinity between this messenger RNA and ribosomal RNA from rabbit liver, and no affinity between this messenger RNA and ribosomal RNA from guinea pig.
Während der Proteinbiosynthese wechselwirken das 3′-Ende der Aminoacyl-tRNA (aatRNA) und der Peptidyl-tRNA spezifisch mit Makromolekülen des Proteinbiosynthese-Apparats. Das 3′-Ende der tRNAs besteht aus einem invarianten C-C-A-Einzelstrang. Die Wechselwirkung des 3′-Endes der aa-tRNA mit dem Elongationsfaktor (EF) ist wichtig für die Bildung des aa-tRNA·EF-Tu·GTP-Komplexes und, nachdem dieser Komplex an das Ribosom gebunden ist, für die GTP-Hydrolyse. Diesem Vorgang folgt die spezifische Bindung des 3′-Endes der Aminoacyl-tRNA an die Acceptorstelle der ribosomalen Peptidyltransferase. In diesem Aufsatz wird ein Modell vorgestellt, nach welchem die C-C-Nucleotide des 3′-Endes der Aminoacyl-tRNA mit einer spezifischen G-G-Sequenz der ribosomalen 23S-RNA Watson-Crick-Basenpaare bilden. Ähnlich bindet die Peptidyl-tRNA mit ihrem 3′-Ende an die komplementäre Sequenz der ribosomalen 23S-RNA. Wir schlagen vor, daß die Bildung der Peptidbindung zwischen den beiden tRNAs durch einen Bereich der 23S-RNA katalysiert wird, der sich in der Nähe des 3′-Endes der Aminoacyl- und der Peptidyl-tRNA befindet. An der Bindung der 3′-Enden der beiden tRNAs sowie an der Katalyse sind zwei Schleifen der 23S-RNA beteiligt, welche durch Faltung in unmittelbare Nachbarschaft gebracht werden können. Das vorgeschlagene Modell setzt eine dynamische Struktur der ribosomalen RNA voraus, die durch Wechselwirkungen mit Elongationsfaktoren und ribosomalen Proteinen funktionell verändert und gesteuert wird.
In the course of protein biosynthesis, the 3′-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3′-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3′-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA·EF-Tu·GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3′-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the CC sequence of the aminoacyl-tRNA 3′-end with a specific GG sequence of the ribosomal 23S RNA. Similarly, peptidyl-tRNA binds with its 3′-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23S RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23S RNA located near the 3′-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23S RNA, brought into close proximity via folding, are involved both in binding the 3′-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.
Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes
mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement
of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond
formation on the ribosome.
Protein synthesis has been studied for more than half a century, but the atomic details of this complex system, the ribosome, began to be revealed in 1999 and 2000 through crystallographic structure determinations. As with all biomolecular systems, the advent of atomic structures are milestones in the process of relating function to structure. During these 10 years there has been an avalanche in biochemical investigations related to the crystal structures as well as new structures pinpointing specific functional details. Three of the central workers in the field (Venkatraman Ramakrishnan, Thomas Steitz and Ada Yonath) were awarded the Nobel Prize for Chemistry 2009. Even though their work has led to significant leaps in understanding, much remains to be clarified. This review will summarize some of the work so far and the understanding achieved.
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