Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.     

Making sense of mimic in translation termination

The mechanism of translation termination has long been a puzzle. Recent crystallographic evidence suggests that the eukaryotic release factor (eRF1), the bacterial release factor (RF2) and the ribosome recycling factor (RRF) all mimic a tRNA structure, whereas biochemical and genetic evidence supports the idea of a tripeptide 'anticodon' in bacterial release factors RF1 and RF2. However, the suggested structural mimicry of RF2 is not in agreement with the tripeptide 'anticodon' hypothesis and, furthermore, recently determined structures using cryo-electron microscopy show that, when bound to the ribosome, RF2 has a conformation that is distinct from the RF2 crystal structure. In addition, hydroxyl-radical probings of RRF on the ribosome are not in agreement with the simple idea that RRF mimics tRNA in the ribosome A-site. All of this evidence seriously questions the simple concept of structural mimicry between proteins and RNA and, thus, leaves only functional mimicry of protein factors of translation to be investigated.     

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.     

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.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Mitochondrial initiation factor 2 of Trypanosoma brucei binds imported formylated elongator-type tRNA(Met)

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA(Met) present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA(Met) carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA(Met). In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA(Met). Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA(Met), suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.     

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.     

Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.     

Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation

In this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with ShineDalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (6570 degrees C) and without the aid of initiator tRNA (tRNAi) or any factor. 'Toeprinting' assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5'-untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codonanticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD-dependent initiation, resembling the pathway prevalent in present-day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas 'leaderless' initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.     

Novel roles for classical factors at the interface between translation termination and initiation.

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.     

Evolution of translational initiation: new insights from the archaea

Recent in silico and experimental data have shed new light on the mechanism and components of translational initiation in archaea. The available data about the structure of archaeal mRNAs, mRNA/ribosome interaction and archaeal translation initiation factors are reviewed and analyzed in the conceptual framework of the evolution of translational initiation. A model of the initiation step of translation in the Last Universal Common Ancestor of extant cells is presented and discussed.     

Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing

Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E. coli RRF constrains it to a well-defined location in the subunit interface cavity. Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry.     

How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.     

Coupling of translation initiation and termination does not depend on the mode of initiation

Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.     

Functional importance of RNA interactions in selection of translation initiation codons

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis -acting mRNA elements and trans -acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.     

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation inEscherichia coli

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribinglacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes. This observation affects the interpretation of many results obtained by use of reporter genes, because the expression from such genes is strongly influenced by the general physiology of the cell.     

Kinetic checkpoint at a late step in translation initiation

The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.