Dynamics of the translational machinery

The recent growth in single molecule studies of translation has provided an insight into the molecular mechanism of ribosomal function. Single molecule fluorescence approaches allowed direct observation of the structural rearrangements occurring during translation and revealed dynamic motions of the ribosome and its ligands. These studies demonstrated how ligand binding affects dynamics of the ribosome, and the role of the conformational sampling in large-scale rearrangements intrinsic to translation elongation. The application of time-resolved cryo-electron microscopy revealed new conformational intermediates during back-translocation providing an insight into ribosomal dynamics from an alternative perspective. Recent developments permitted examination of conformational and compositional dynamics of the ribosome in real-time through multiple cycles of elongation at the single molecule level. The zero-mode waveguide approach allowed direct observation of the compositional dynamics of tRNA occupancy on the elongating ribosome. The emergence of single molecule in vivo techniques provided insights into the mechanism and regulation of translation at the organismal level.     

A paradigm for ribosome fidelity modulation in protein synthesis

The ribosome, an integral component of the protein synthetic apparatus, plays a role in mediating the fidelity of the translation of genetic information. It is proposed here that ribosome fidelity modulation can be achieved in part through ribosomal RNA interactions with messenger RNA. These nucleic acid interactions generate alterations in the configuration of the ribosome which enhance the particles affinity for classes on specific transfer RNAs. These changes are directly related to the information content of a given messenger RNA which is being translated and yield an increase in the fidelity of translation. The molecular mechanism for effecting the ribosomal structural changes in response to the genetic code is described and correlated with the properties of ribosomal proteins and transfer RNAs.     

A model for the study of ligand binding to the ribosomal RNA helix h44

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand–RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.     

Translation initiation: structures, mechanisms and evolution

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.     

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.     

Engaging the ribosome: universal IFs of translation

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.     

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.     

The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.     

Structure of the chloroplast ribosome: novel domains for translation regulation

Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 Å. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.     

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.     

Binding of the IRES of hepatitis C virus RNA to the 40S ribosomal subunit: Role of p40

Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to 40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA.     

A decade of progress in understanding the structural basis of protein synthesis

The key reaction of protein synthesis, peptidyl transfer, is catalysed in all living organisms by the ribosome - an advanced and highly efficient molecular machine. During the last decade extensive X-ray crystallographic and NMR studies of the three-dimensional structure of ribosomal proteins, ribosomal RNA components and their complexes with ribosomal proteins, and of several translation factors in different functional states have taken us to a new level of understanding of the mechanism of function of the protein synthesis machinery. Among the new remarkable features revealed by structural studies, is the mimicry of the tRNA molecule by elongation factor G, ribosomal recycling factor and the eukaryotic release factor 1. Several other translation factors, for which three-dimensional structures are not yet known, are also expected to show some form of tRNA mimicry. The efforts of several crystallographic and biochemical groups have resulted in the determination by X-ray crystallography of the structures of the 30S and 50S subunits at moderate resolution, and of the structure of the 70S subunit both by X-ray crystallography and cryo-electron microscopy (EM). In addition, low resolution cryo-EM models of the ribosome with different translation factors and tRNA have been obtained. The new ribosomal models allowed for the first time a clear identification of the functional centres of the ribosome and of the binding sites for tRNA and ribosomal proteins with known three-dimensional structure. The new structural data have opened a way for the design of new experiments aimed at deeper understanding at an atomic level of the dynamics of the system.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.     

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

Toward an understanding of the structural basis of translation

The recently solved X-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.     

RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

Single-molecule structural dynamics of EF-G--ribosome interaction during translocation

EF-G catalyzes translocation of mRNA and tRNAs within the ribosome during protein synthesis. Detection of structural states in the reaction sequence that are not highly populated can be facilitated by studying the process one molecule at a time. Here we present single-molecule studies of translocation showing that, for ribosomes engaged in poly(Phe) synthesis, fluorescence resonance energy transfer (FRET) between the G' domain of EF-G and the N-terminal domain of ribosomal protein L11 occurs within two rapidly interconverting states, having FRET efficiencies of 0.3 and 0.6. The antibiotic fusidic acid increases the population of the 0.6 state, indicating that it traps the ribosome.EF-G complex in a preexisting conformation formed during translation. Only the 0.3 state is observed when poly(Phe) synthesis is prevented by omission of EF-Tu, or in studies on vacant ribosomes. These results suggest that the 0.6 state results from the conformational lability of unlocked ribosomes formed during translocation. An idling state, possibly pertinent to regulation of protein synthesis, is detected in some ribosomes in the poly(Phe) system.     

GTPases of prokaryotic translational apparatus

Four protein factors, belonging to the GTPase superfamily, participate in bacterial biosynthesis: IF2, EF-G, EF-Tu and RF3. The exact role and mechanism of action of these proteins was of particular interest over the last several decades. Recent advances in structural methods of ribosomal research, especially application of cryoelectron microscopy, provided powerful experimental tools for the investigation of ribosomal dynamics during translation. Simultaneously, progress in the biochemical investigation of translation allowed us to link structural rearrangements occurring in the ribosome to functional changes in the ribosome-bound translational GTPases--GDP/GTP exchange, GTPase activation and its conformational changes. Accumulated data have lead to formulation of current models of mechanisms of translation. More and more facts testify in favor of the idea that the ribosome plays a prominent role both in the nucleotide exchange and in GTPase activation, thus playing the role both of GAP and GEF for RF3, IF2 and EF-G. In our work we attempted to systematize the most important experimental findings and models for mechanisms of GTPases function and regulation in prokaryotic translation.