The structure and function of initiation factors in eukaryotic protein synthesis

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.     

Yeast as a sensor of factors affecting the accuracy of protein synthesis

The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination. Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression. All of these events produce incorrect protein products. Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery. Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors). The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis. This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis.Received 27 November 2002; received after revision 16 April 2003; accepted 25 April 2003     

Structural studies of elongation and release factors

The elongation and termination steps of protein synthesis are controlled by elongation and release factors, respectively. Elongation factors deliver the aminoacyl tRNA to the ribosomal A site, ensuring the elongation of the nascent polypeptide chain by one amino acid at a time, while release factors recognize the stop codons and trigger the release of the polypeptide from the ribosome. Recently, highresolution crystal structures of ribosomes as well as translation factors on and off the ribosome have contributed a great deal to our understanding of the molecular basis of protein synthesis. This review concentrates on recent developments in our understanding of the elongation and termination steps of protein synthesis, particularly the roles of translation factors and their similarities and differences in the eukaryotic cytosol and prokaryotic systems, through a combination of structural and biochemical studies. Received 25 October 2007; received after revision 5 December 2007; accepted 7 December 2007     

The E-site story: the importance of maintaining two tRNAs on the ribosome during protein synthesis

In the sixties James Watson suggested a twosite model for the ribosome comprising the P site for the peptidyl transfer RNA (tRNA) before peptide-bond formation and the A site, where decoding takes place according to the codon exposed there. In the eighties a third tRNA binding site was detected, the E site, which was specific for deacylated tRNA and turned out to be a universal feature of ribosomes. However, despite having three tRNA binding sites, only two tRNAs occupy the ribosome at a time during protein synthesis: at the A and P sites before translocation (PRE state) and at the P and E sites after translocation (POST state). The importance of having two tRNAs in the POST state has been revealed during the last 25 years, showing that the E site contributes two fundamental features: (i) the fact that incorporation of a wrong amino acid is not harmful for the cell (only 1 in about 400 misincorporations destroys the function of a protein) stems from the presence of an E-tRNA; (ii) maintenance of the reading frame is one of the most remarkable achievements of the ribosome, essential for faithful translation of the genetic information. The presence of the POST state E-tRNA prevents loss of the reading frame. Received 14 March 2006; received after revision 8 June 2006; accepted 4 August 2006     

More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5′ end of the mRNA and scans the 5′ untranslated region (5′UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5′UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N ⁶-methyladenosine (m⁶A) residues in their 5′UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.     

The accuracy of aminoacylation--ensuring the fidelity of the genetic code

The fidelity of protein biosynthesis rests not only on the proper interaction of the messenger RNA codon with the anticodon of the tRNA, but also on the correct attachment of amino acids to their corresponding (cognate) transfer RNA (tRNA) species. This process is catalyzed by the aminoacyl-tRNA synthetases which discriminate with remarkable selectivity amongst many structurally similar tRNAs. The basis for this highly specific recognition of tRNA by these enzymes (also referred to as 'tRNA identity') is currently being elucidated by genetic, biochemical and biophysical techniques. At least two factors are important in determining the accuracy of aminoacylation: a) 'identity elements' in tRNA denote nucleotides in certain positions crucial for protein interactions determining specificity, and b) the occurrence in vivo of competition between synthetases for a particular tRNA which may have ambiguous identity.     

eIF2A,an initiator tRNA carrier refractory to eIF2α kinases,functions synergistically with eIF5B

The initiator tRNA (Met-tRNA _i ^Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA _i ^Met on the small ribosomal subunit. Eukarya contain the Met-tRNA _i ^Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA _i ^Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA _i ^Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B–eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.     

Isolation and partial characterization of receptor sites of normal human lymphocytes for lectins]

Glycoproteins of the lymphocyte surface are involved in many membrane mediated events. Their specific carbohydrate determinants might interact with lectins. Purification of macromolecules released from normal human lymphocytes by trypsin was performed using gel filtration and affinity properties for Ricinus sanguineus agglutinin. Some structural and biochemical characteristics are given. Relation to HLA determinants and surface immunoglobulins is discussed.     

Novel features in the tRNA-like world of plant viral RNAs

tRNA-like domains are found at the 3' end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence similarities with tRNA are those found in valine accepting structures from tymoviruses (e.g. TYMV). All the viral tRNA mimics present a pseudoknotted amino acid accepting stem, which confers special structural and functional characteristics. In this review emphasis is given to newly discovered tRNA-like structures (e.g. in furoviruses) and to recent advances in the understanding of their three-dimensional architecture, which mimics L-shaped tRNA. Identity determinants in tRNA-like domains for aminoacylation are described, and evidence for their functional expression, as in tRNAs, is given. Properties of engineered tRNA-like domains are discussed, and other functional mimicries with tRNA are described (e.g. interaction with elongation factors and tRNA maturation enzymes). A final section reviews the biological role of the tRNA-like domains in amplification of viral genomes. In this process, in which the mechanisms can vary in specificity and efficiency according to the viral genus, function can be dependent on the aminoacylation properties of the tRNA-like domains and/or on structural properties within or outside these domains.     

Structural determinants of protein folding

The last several decades have seen an explosion of knowledge in the field of structural biology. With critical advances in spectroscopic techniques in examining structures of biomacromolecules, in maturation of molecular biology techniques, as well as vast improvements in computation prowess, protein structures are now being elucidated at an unprecedented rate. In spite of all the recent advances, the protein folding puzzle remains as one of the fundamental biochemical challenges. A facet to this empiric problem is the structural determinants of protein folding. What are the driving forces that pivot a polypeptide chain to a specific conformation amongst the vast conformation space? In this review, we shall discuss some of the structural determinants to protein folding that have been identified in the recent decades.     

What's new in translation initiation? The first translation determines the fate of mRNA

The two terms 'translation' and 'protein synthesis' are interchangeable in describing the process whereby the genetic code in the form of messenger RNA (mRNA) is deciphered such that amino acids cognate with the triplet code are joined end to end to form a peptide chain. However, new data suggest that the initial act of translation on newly synthesised mRNA also functions to proofread mRNA for errors. Aberrant mRNAs detected in this way are rapidly degraded before their encoded proteins impede normal cell function. Initiation of surveillance translation appears to differ from that of regular protein synthesis in three ways: (i) composition of the substrate; (ii) temporal and spatial restrictions; (iii) factors used to recruit the ribosome. This review discusses translational aspects of mRNA surveillance, primarily in the context of the mammalian system, although much information has come from studies in yeast and other organisms.     

Diversity and roles of (t)RNA ligases

The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases.     

Misacylation of tRNA in prokaryotes: a re-evaluation

Misacylation of tRNA by a non-cognate amino acid is a natural phenomenon and occurs with a frequency of approximately 1 in 10,000 due to occasional mistakes in aminoacyl transfer RNA (tRNA) synthesis. In a number of prokaryotic organisms, misacylation of selenocysteinyl tRNA, glutaminyl tRNA and aspartyl tRNAs has particular physiological meaning. Recently, misacylation has emerged as a powerful tool for studying specific interactions between aa-tRNAs and associated protein factors. The present review provides an overview of the application of misacylated tRNA in research. Received 27 April 2005; received after revision 2 November 2005; accepted 5 December 2005     

Prokaryotic genetic code

The prokaryotic genetic code has been influenced by directional mutation pressure (GC/AT pressure) that has been exerted on the entire genome. This pressure affects the synonymous codon choice, the amino acid composition of proteins and tRNA anticodons. Unassigned codons would have been produced in bacteria with extremely high GC or AT genomes by deleting certain codons and the corresponding tRNAs. A high AT pressure together with genomic economization led to a change in assignment of the UGA codon, from stop to tryptophan, in Mycoplasma.     

Expression of the mutant gene for L-gulono-gamma-lactone oxidase in scurvy-prone rats

A mutant strain of Wistar rats with L-gulono-gamma-lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono-gamma-lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)+RNA from the liver of the mutant rat was found to contain an L-gulono-gamma-lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono-gamma-lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.     

Protein folding: concepts and perspectives

In this review, the main concepts of protein folding, as deduced from both theoretical and experimental in vitro studies, are presented. The thermodynamic aspects from Anfinsen's postulate, Levinthal's paradox to the concept of folding funnel as proposed by Wolynes and coworkers are described. Concerning the folding pathway(s), particular attention is brought to bear on the early steps that initiate the process in the light of the results of the fast and even ultrafast techniques presently being used. The role of structural domains as folding units is discussed. Last, from the recent studies, it can be concluded that the main rules deduced from the in vitro folding studies are valid for the folding of a nascent polypeptide chain in vivo.     

tRNA structural and functional changes induced by oxidative stress

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level.     

Translation control of UCP2 synthesis by the upstream open reading frame

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane. In vivo low expression of UCP2 contrasts with a high UCP2 mRNA level, and induction of UCP2 expression occurs without change in mRNA level, demonstrating a translational control. The UCP2 mRNA is characterized by a long 5′ untranslated region (5′UTR), in which an upstream open reading frame (uORF) codes for a 36-amino-acid sequence. The 5′UTR and uORF have an inhibitory role in the translation of UCP2. The present study demonstrates that the 3′ region of the uORF is a major determinant for this inhibitory role. In this 3′ region, a single-base substitution that kept the codon sense unchanged significantly modified UCP2 translation, whereas some important amino acid changes had no effect. We discuss our results within the framework of the existing models explaining initiation of translation downstream of a uORF. Received 22 March 2006; received after revision 19 May 2006; accepted 8 June 2006 C. Hurtaud and C. Gelly contributed equally to this work.