Mitotic modulation of translation elongation factor 1 leads to hindered tRNA delivery to ribosomes

Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.     

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.     

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.     

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3

The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3). eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site. To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene. The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities. In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A. Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A. In addition, this mutant form of eEF1A shows reduced binding of eEF3. Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis.     

Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.     

Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis through Reduced ATPase Activity

Translation elongation is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic elongation factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome, whereas eEF2 (EF-G in bacteria) translocates the ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP binding cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7d region of the chromodomain-like insertion. Specific mutations in this domain result in reduced growth rate as well as slower translation elongation. In vitro analysis demonstrates that these mutations do not affect the ability of eEF3 to interact with the ribosome. Kinetic analysis revealed a larger turnover number for ribosomes in comparison to eEF3, indicating that the partial reactions involving the ribosome are significantly faster than that of eEF3. Mutations in the chromodomain-like insertion severely compromise the ribosome stimulated ATPase of eEF3, strongly suggesting that it exerts an allosteric effect on the hydrolytic activity of eEF3. The chromodomain-like insertion is, therefore, vital to eEF3 function and may be targeted for developing novel antifungal drugs.     

Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance

Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased -1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.     

Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES

Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.     

Structural Insights into the Role of Diphthamide on Elongation Factor 2 in mRNA Reading-Frame Maintenance

One of the most critical steps of protein biosynthesis is the coupled movement of mRNA, which encodes genetic information, with tRNAs on the ribosome. In eukaryotes, this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-electron microscopy structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights into the role of diphthamide in the mechanism of translation fidelity in eukaryotes.     

Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the alpha subunit of human eIF2 (heIF2alpha). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an alpha-helical subdomain. The C-terminal domain adopts an alphabeta-fold very similar to the C-terminal domain of elongation factor (eEF) 1Balpha, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2alpha/eIF2gamma and eEF1Balpha/eEF1A suggest a model for the interaction of eIF2alpha with eIF2gamma, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2alpha/gamma from the functionally related elongation factor eEF1Balpha/eEF1A complex.     

Ribosomal RNA is the target for oxazolidinones, a novel class of translational inhibitors.

Oxazolidinones are antibacterial agents that act primarily against gram-positive bacteria by inhibiting protein synthesis. The binding of oxazolidinones to 70S ribosomes from Escherichia coli was studied by both UV-induced cross-linking using an azido derivative of oxazolidinone and chemical footprinting using dimethyl sulphate. Oxazolidinone binding sites were found on both 30S and 50S subunits, rRNA being the only target. On 16S rRNA, an oxazolidinone footprint was found at A864 in the central domain. 23S rRNA residues involved in oxazolidinone binding were U2113, A2114, U2118, A2119, and C2153, all in domain V. This region is close to the binding site of protein L1 and of the 3' end of tRNA in the E site. The mechanism of action of oxazolidinones in vitro was examined in a purified translation system from E. coli using natural mRNA. The rate of elongation reaction of translation was decreased, most probably because of an inhibition of tRNA translocation, and the length of nascent peptide chains was strongly reduced. Both binding sites and mode of action of oxazolidinones are unique among the antibiotics known to act on the ribosome.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.     

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.     

Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.     

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3’s known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.     

Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.     

GTPases of the Translation Apparatus

Protein biosynthesis is a complex biochemical process involving a number of stages at which different translation factors specifically interact with ribosome. Some of these factors belong to GTP-binding proteins, or G-proteins. Due to their functioning, GTP is hydrolyzed to yield GDP and the inorganic phosphate ion P_i. Interaction with ribosome enhances GTPase activity of translation factors; i.e., ribosome plays a role of GTPase-activating protein (GAP). GTPases involved in translation interact with ribosome at every stage of protein biosynthesis. Initiation factor 2 (IF2) catalyzes initiator tRNA binding to the ribosome P site and subsequent binding of the 50S subunit to the initiation complex of the 30S subunit. Elongation factor Tu (EF-Tu) controls aminoacyl-tRNA delivery to the ribosome A site, while elongation factor G (EF-G) catalyzes translocation of the mRNA-tRNA complex by one codon on the ribosome. Release factor 3 (RF3) catalyzes the release of termination factors 1 or 2 (RF1 or RF2) from the ribosomal complex after completion of protein synthesis and peptidyl-tRNA hydrolysis. The functional properties of translational GTPases as related to other G-proteins, the putative mechanism of GTP hydrolysis, structural features, and the functional cycles of translational GTPases are considered.     

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.     

Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.