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.     

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.     

The metaphorical swiss army knife: The multitude and diverse roles of HEAT domains in eukaryotic translation initiation

Biomolecular associations forged by specific interaction among structural scaffolds are fundamental to the control and regulation of cell processes. One such structural architecture, characterized by HEAT repeats, is involved in a multitude of cellular processes, including intracellular transport, signaling, and protein synthesis. Here, we review the multitude and versatility of HEAT domains in the regulation of mRNA translation initiation. Structural and cellular biology approaches, as well as several biophysical studies, have revealed that a number of HEAT domain-mediated interactions with a host of protein factors and RNAs coordinate translation initiation. We describe the basic structural architecture of HEAT domains and briefly introduce examples of the cellular processes they dictate, including nuclear transport by importin and RNA degradation. We then focus on proteins in the translation initiation system featuring HEAT domains, specifically the HEAT domains of eIF4G, DAP5, eIF5, and eIF2Bϵ. Comparative analysis of their remarkably versatile interactions, including protein–protein and protein–RNA recognition, reveal the functional importance of flexible regions within these HEAT domains. Here we outline how HEAT domains orchestrate fundamental aspects of translation initiation and highlight open mechanistic questions in the area.     

A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.     

依赖于5′端非编码区高级结构的真核生物mRNA翻译调控

翻译水平的调控是真核基因表达调控的重要环节.近年来的研究表明,许多真核基因的翻译依赖于RNA 5′端非编码区的结构元件.一些小结构元件,如铁离子反应元件,具有1个茎环结构,由铁离子介导控制转铁蛋白的翻译. 核糖开关通过结合特定代谢分子在2种结构状态下切换,调控可变剪接和翻译起始.另1个高度结构化的mRNA元件是内部核糖体进入位点,通过富集核糖体和起始因子促进基因的表达.本文综述了依赖于小结构元件、内部核糖体进入位点和核糖开关的真核基因翻译起始调控相应的研究成果和研究方法.对于研究的前景以及可能存在的挑战也作出阐述.     

HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.     

Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome

Protein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.     

L-Myc protein synthesis is initiated by internal ribosome entry

An internal ribosome entry segment (IRES) has been identified in the 5' untranslated region (5' UTR) of two members of the myc family of proto-oncogenes, c-myc and N-myc. Hence, the synthesis of c-Myc and N-Myc polypeptides can involve the alternative mechanism of internal initiation. Here, we show that the 5' UTR of L-myc, another myc family member, also contains an IRES. Previous studies have shown that the translation of mRNAs containing the c-myc and N-myc IRESs can involve both cap-dependent initiation and internal initiation. In contrast, the data presented here suggest that internal initiation can account for all of the translation initiation that occurs on an mRNA with the L-myc IRES in its 5' UTR. Like many other cellular IRESs, the L-myc IRES appears to be modular in nature and the entire 5' UTR is required for maximum IRES efficiency. The ribosome entry window within the L-myc IRES is located some distance upstream of the initiation codon, and thus, this IRES uses a "land and scan" mechanism to initiate translation. Finally, we have derived a secondary structural model for the IRES. The model confirms that the L-myc IRES is highly structured and predicts that a pseudoknot may form near the 5' end of the mRNA.     

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.     

Site-Directed Mutagenesis of a Saccharomyces Cerevisiae Mitochondrial Translation Initiation Codon

We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.     

eIF5A binds to translational machinery components and affects translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archebacteria to mammals. Although this protein was originally identified as a translation initiation factor, subsequent experiments did not support a role for eIF5A in general translation. In this work, we demonstrate that eIF-5A interacts with structural components of the 80S ribosome, as well as with the translation elongation factor 2 (eEF2). Moreover, eIF5A is further shown to cofractionate with monosomes in a translation-dependent manner. Finally, eIF5A mutants show altered polysome profiles and are sensitive to translation inhibitors. Our results re-establish a function for eIF5A in translation and suggest a role for this factor in translation elongation instead of translation initiation.     

Structural basis for the control of translation initiation during stress

During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures. Furthermore, protein Y competes with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature.     

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites

Background  

Utilization of alternative initiation sites for protein translation directed by non-AUG codons in mammalian mRNAs is observed with increasing frequency. Alternative initiation sites are utilized for the synthesis of important regulatory proteins that control distinct biological functions. It is, therefore, of high significance to define the parameters that allow accurate bioinformatic prediction of alternative translation initiation sites (aTIS). This study has investigated 5'-UTR regions of mRNAs to define consensus sequence properties and structural features that allow identification of alternative initiation sites for protein translation.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

The different pathways of HIV genomic RNA translation

Lentiviruses, the prototype of which is HIV-1, can initiate translation either by the classical cap-dependent mechanism or by internal recruitment of the ribosome through RNA domains called IRESs (internal ribosome entry sites). Depending on the virus considered, the mechanism of IRES-dependent translation differs widely. It can occur by direct binding of the 40S subunit to the mRNA, necessitating a subset or most of the canonical initiation factors and/or ITAF (IRES trans-acting factors). Nonetheless, a common feature of IRESs is that ribosomal recruitment relies, at least in part, on IRES structural determinants. Lentiviral genomic RNAs present an additional level of complexity, as, in addition to the 5'-UTR (untranslated region) IRES, the presence of a new type of IRES, embedded within Gag coding region was described recently. This IRES, conserved in all three lentiviruses examined, presents conserved structural motifs that are crucial for its activity, thus reinforcing the link between RNA structure and function. However, there are still important gaps in our understanding of the molecular mechanism underlying IRES-dependent translation initiation of HIV, including the determination of the initiation factors required, the dynamics of initiation complex assembly and the dynamics of the RNA structure during initiation complex formation. Finally, the ability of HIV genomic RNA to initiate translation through different pathways questions the possible mechanisms of regulation and their correlation to the viral paradigm, i.e. translation versus encapsidation of its genomic RNA.     

Regulation of protein synthesis by mRNA structure

In addition to the m⁷G cap structure, the length of the 5 UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structuresper se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.     

Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.     

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.     

Caliciviruses differ in their functional requirements for eIF4F components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.