The busiest of all ribosomal assistants: elongation factor Tu

During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review.     

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6?? resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.     

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.     

Mechanistic Role of Structurally Dynamic Regions in Dicistroviridae IGR IRESs

Dicistroviridae intergenic region (IGR) internal ribosome entry site(s) (IRES) RNAs drive a cap-independent pathway of translation initiation, recruiting both small and large ribosomal subunits to viral RNA without the use of any canonical translation initiation factors. This ability is conferred by the folded three-dimensional structure of the IRES RNA, which has been solved by X-ray crystallography. Here, we report the chemical probing of Plautia stali intestine virus IGR IRES in the unbound form, in the 40S-subunit-bound form, and in the 80S-ribosome-bound form. The results, when combined with an analysis of crystal structures, suggest that parts of the IRES RNA change structure as the preinitiation complex forms. Using mutagenesis coupled with native gel electrophoresis, preinitiation complex assembly assays, and translation initiation assays, we show that these potentially structurally dynamic elements of the IRES are involved in different steps in the pathway of ribosome recruitment and translation initiation. Like tRNAs, it appears that the IGR IRES undergoes local structural changes that are coordinated with structural changes in the ribosome, and these are critical for the IRES mechanism of action.     

Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.     

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.     

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.     

Structural insights on the translation initiation complex: ghosts of a universal initiation complex

All living organisms utilize ribosomes to translate messenger RNA into proteins. Initiation of translation, the process of bringing together mRNA, initiator transfer RNA, and the ribosome, is therefore of critical importance to all living things. Two protein factors, IF1 (a/eIF1A) and IF2 (a/eIF5B), are conserved among all three kingdoms of life and have been called universal initiation factors (Roll-Mecak et al., 2001). Recent X-ray, NMR and cryo-EM structures of the universal factors, alone and in complex with eubacterial ribosomes, point to the structural homology among the initiation factors and initiation complexes. Taken together with genomic and functional evidence, the structural studies allow us to predict some features of eukaryotic and archaeal initiation complexes. Although initiation of translation in eukaryotes and archaea requires more initiation factors than in eubacteria we propose the existence of a common denominator initiation complex with structural and functional homology across all kingdoms of life.     

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.     

Novel G-protein complex whose requirement is linked to the translational status of the cell

G proteins, which bind and hydrolyze GTP, are involved in regulating a variety of critical cellular processes, including the process of protein synthesis. Many members of the subfamily of elongation factor class G proteins interact with the ribosome and function to regulate discrete steps during the process of protein synthesis. Despite sequence similarity to factors involved in translation, a role for the yeast Hbs1 protein has not been defined. In this work we have identified a genetic relationship between genes encoding components of the translational apparatus and HBS1. HBS1, while not essential for viability, is important for efficient growth and protein synthesis under conditions of limiting translation initiation. The identification of an Hbs1p-interacting factor, Dom34p, which shares a similar genetic relationship with components of the translational apparatus, suggests that Hbs1p and Dom34p may function as part of a complex that facilitates gene expression. Dom34p contains an RNA binding motif present in several ribosomal proteins and factors that regulate translation of specific mRNAs. Thus, Hbs1p and Dom34p may function together to help directly or indirectly facilitate the expression either of specific mRNAs or under certain cellular conditions.     

Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.     

GTPases of translational apparatus

Protein biosynthesis is a complex biochemical process. It integrates multiple steps where different translation factors specifically interact with the ribosome in a precisely defined order. Among the translation factors one can find multiple GTP-binding or G-proteins. Their functioning is accompanied by GTP hydrolysis to the GDP and inorganic phosphate ion Pi. Ribosome stimulates the GTPase activity of the translation factors, thus playing a role analogues to GTPase-activating proteins (GAP). Translation factors--GTPases interact with the ribosome at all stages of protein biosynthesis. Initiation factor 2 (IF2) catalyse initiator tRNA binding to the ribosomal P-site and subsequent subunit joining. Elongation factor Tu (EF-Tu) is responsible for the aminoacyl-tRNA binding to the ribosomal A-site, while elongation factor G (EF-G) catalyses translocation of mRNA in the ribosome by one codon, accompanied by tRNA movement between the binding sites. In its turn, release factor 3 (RF3) catalyse dissociation of the ribosomal complex with release factors 1 or 2 (RF1 or RF2) following the peptide release. This review is devoted to the functional peculiarities of translational GTPases as related to other G-proteins. Particularly, to the putative GTPase activation mechanism, structure and functional cycles.     

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.     

In Vitro Molecular Characterization of RNA–Proteins Interactions During Initiation of Translation of a Wild-Type and a Mutant Coxsackievirus B3 RNAs

Translation initiation of Coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5′ untranslated region. Host cell factors involved in this process include some canonical translation factors and additional RNA-binding proteins. We have, previously, described that the Sabin3-like mutation (U475 → C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. With the aim to identify proteins interacting with CVB3 wild-type and Sabin3-like IRESes and to study interactions between HeLa cell or BHK-21 protein extracts and CVB3 RNAs, UV-cross-linking assays were performed. We have observed a number of proteins that specifically interact with both RNAs. In particular, molecular weights of five of these proteins resemble to those of the eukaryotic translation initiation factors 4G, 3b, 4B, and PTB. According to cross-linking patterns obtained, we have demonstrated a better affinity of CVB3 RNA binding to BHK-21 proteins and a reduced interaction of the mutant RNA with almost cellular polypeptides compared to the wild-type IRES. On the basis of phylogeny of some initiation factors and on the knowledge of the initiation of translation process, we focused on the interaction of both IRESes with eIF3, p100 (eIF4G), and 40S ribosomal subunit by filter-binding assays. We have demonstrated a better affinity of binding to the wild-type CVB3 IRES. Thus, the reduction efficiency of the mutant RNA to bind to cellular proteins involved in the translation initiation could be the reason behind inefficient IRES function.     

Single-molecule imaging of full protein synthesis by immobilized ribosomes

How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro. Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli. Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosome-polypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding.     

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.