Interchanging Functionality Among Homologous Elongation Factors Using Signatures of Heterotachy

Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.     

Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes.

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tumt, show distinct differences in their affinities for guanine nucleotides and in their interactions with elongation factor Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu binds GDP much more tightly than EF-Tumt. This difference does not reside in domain I alone but is regulated by interactions with domains II and III. All the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRNA although some had an increased or decreased activity in this assay. The activity of E. coli EF-Tu but not of EF-Tumt is stimulated by E. coli EF-Ts. The presence of any one of the domains of EF-Tumt in the prokaryotic factor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tumt allowed the mitochondrial factor to interact with bacterial EF-Ts. This observation indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Tsmt interacts with all of the chimeras produced. However, with the exception of domain III exchanges, it inhibits the activities of the chimeras indicating that it could not be productively released to allow formation of the ternary complex. The unique ability of EF-Tumt to promote binding of mitochondrial Phe-tRNAPhe to the A-site of the ribosome resides in domains I and II. These studies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains.     

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.     

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.     

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.     

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.     

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

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.     

A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome.

Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.     

Mutagenesis of glutamine 290 in Escherichia coli and mitochondrial elongation factor Tu affects interactions with mitochondrial aminoacyl-tRNAs and GTPase activity

Elongation factor Tu (EF-Tu) is responsible for the delivery of the aminoacyl-tRNAs (aa-tRNA) to the ribosome during protein synthesis. The primary sequence of domain II of EF-Tu is highly conserved. However, several residues thought to be important for aa-tRNA binding in this domain are not conserved between the mammalian mitochondrial and bacterial factors. One of these residues is located at position 290 (Escherichia coli numbering). Residue 290 is Gln in most of the prokaryotic factors but is conserved as Leu (L338) in the mammalian mitochondrial factors. This residue is in a loop contacting the switch II region of domain I in the GTP-bound structure. It also helps to form the binding pocket for the 5' end of the aa-tRNA in the ternary complex. In the present work, Leu338 was mutated to Gln (L338Q) in EF-Tu(mt). The complementary mutation was created at the equivalent position in E. coli EF-Tu (Q290L). EF-Tu(mt) L338Q functions as effectively as wild-type EF-Tu(mt) in poly(U)-directed polymerization with both prokaryotic and mitochondrial substrates and in ternary complex formation assays with E. coli aa-tRNA. However, the L338Q mitochondrial variant has a reduced affinity for mitochondrial Phe-tRNA(Phe). E. coli EF-Tu Q290L is more active in poly(U)-directed polymerization with both mitochondrial and prokaryotic substrates and has a higher GTPase activity in both the absence and presence of ribosomes. Surprisingly, while E. coli EF-Tu Q290L is more active in polymerization with mitochondrial Phe-tRNA(Phe), this variant has low activity in the formation of a stable ternary complex with mitochondrial aa-tRNA.     

The interaction between interferon-induced protein with tetratricopeptide repeats-1 and eukaryotic elongation factor-1A

It has been shown previously that in mammalian cells, interferon-induced protein with tetratricopeptide repeats-1(IFIT1) is rapidly synthesized in response to viral infection, functions as an inhibitor of translation by binding to the eukaryotic initiation factor-3, and consequently assigns resistive activity against viral invasion to cells. It has also been reported that IFIT1 is rapidly produced in response to other cell stress agents with no direct relation to virus such as bacterial lipopolysaccharide and interleukin-1, but its function under these non-viral infection cell stress conditions has yet to be elucidated. Here, we demonstrate an interaction between IFIT1 and eukaryotic elongation factor-1A (eEF1A) both in vitro, using recombinant proteins as bait in pull-down assays, and in vivo, using laser confocal microscopy and immunoprecipitation. In addition, we report the initial determination of the domain of IFIT1 that mediates this interaction. We also display that both IFIT1 and eEF1A protein levels are rapidly elevated, prolonged in tumor necrosis factor alpha pre-treated Raw264.7 cells, and most of those cells are induced to death by the end of investigations. Our results imply that under some stressful stimulations IFIT1 may participate in cell death pathways by interaction with eEF1A.     

The eEF1γ subunit contacts RNA polymerase II and binds vimentin promoter region

Here, we show that the eukaryotic translation elongation factor 1 gamma (eEF1γ) physically interacts with the RNA polymerase II (pol II) core subunit 3 (RPB3), both in isolation and in the context of the holo-enzyme. Importantly, eEF1γ has been recently shown to bind Vimentin mRNA. By chromatin immunoprecipitation experiments, we demonstrate, for the first time, that eEF1γ is also physically present on the genomic locus corresponding to the promoter region of human Vimentin gene. The eEF1γ depletion causes the Vimentin protein to be incorrectly compartmentalised and to severely compromise cellular shape and mitochondria localisation. We demonstrate that eEF1γ partially colocalises with the mitochondrial marker Tom20 and that eEF1γ depletion increases mitochondrial superoxide generation as well as the total levels of carbonylated proteins. Finally, we hypothesise that eEF1γ, in addition to its role in translation elongation complex, is involved in regulating Vimentin gene by contacting both pol II and the Vimentin promoter region and then shuttling/nursing the Vimentin mRNA from its gene locus to its appropriate cellular compartment for translation.     

Chaperone-like activity of mammalian elongation factor eEF1A: renaturation of aminoacyl-tRNA synthetases

Eukaryotic translational elongation factor eEF1A is known to be responsible for the binding of codon-specific aminoacyl-tRNAs to the ribosome. In this study, we report that in addition to this canonical function, eEF1A is able to promote the renaturation of aminoacyl-tRNA synthetases (ARS) and protect them against denaturation by dilution. The full recovery of the phenylalanyl- (PheRS) and seryl-tRNA synthetase (SerRS) activities was achieved in the presence of 4 microM eEF1A, while bovine serum albumin at similar concentration had no renaturation effect. Remarkably, in vitro renaturation occurs at the molar ratio of eEF1A to ARS equivalent to that found in the cytoplasm of higher eukaryotic cells. The eEF1A.GDP and eEF1A.GTP complexes were shown to be similar in their effect on the phenylalanyl-tRNA synthetase renaturation. Thus, we conclude that the chaperone-like activity of eEF1A might be important for maintaining the enzymes activity in the protein synthesis compartments of mammalian cells.     

Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes

The mammalian mitochondrial complex consisting of elongation factors EF-Tu and EF-Ts (EF-Tu.Tsmt) is capable of efficiently binding aminoacyl-tRNA to the ribosome in the presence and absence of guanine nucleotides. In the presence of GTP the binding reaction is catalytic. In the absence of guanine nucleotides, or in the presence of a non-hydrolyzable GTP analog, only one round of ribosome binding occurs. EF-Tu.Tsmt is capable of forming a ternary complex with GTP and Escherichia coli Phe-tRNA as demonstrated by gel filtration chromatography, nitrocellulose filter binding, and by protection of the aminoacyl-tRNA bond from hydrolysis. GDP and the non-hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate are also capable of facilitating ternary complex formation with EF-Tu.Tsmt, but are less effective. No kinetic advantage results from the formation of this ternary complex prior to ribosome binding, and EF-Tu.Tsmt may actually bind aminoacyl-tRNA directly to the ribosome prior to binding GTP. These results suggest that a variation of the prokaryotic elongation cycle is occurring in animal mitochondria. N-Ethylmaleimide inhibits the activity of EF-Tu.Tsmt in polymerization and in ribosome binding. However, the activity of the EF-Tsmt which can be measured independently, is not altered.     

The elongation, termination, and recycling phases of translation in eukaryotes

This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.     

Solution structure of the 162 residue C-terminal domain of human elongation factor 1Bgamma

The multisubunit elongation factor 1 (eEF1) is required for the elongation step of eukaryotic protein synthesis. The eEF1 complex consists of four subunits: eEF1A, a G-protein that shuttles aminoacylated tRNAs to the ribosome; eEF1Balpha and eEF1Bbeta, two guanine nucleotide exchange factors, and eEF1Bgamma. Although its exact function remains unknown, this latter subunit is present in all eukaryotes. Recombinant human eEF1Bgamma has been purified and shown to consist of two independent domains. We have utilized high resolution NMR to determine the three-dimensional structure of the 19 kDa C-terminal fragment (domain 2). The structure consists of a five-stranded anti-parallel beta-sheet surrounded by alpha-helices and resembles a contact lens. Highly conserved residues are mainly located on the concave face, suggesting thereby that this side of the molecule might be involved in some biologically relevant interface(s). Although the isolated domain 2 appears to be mostly monomeric in solution, biochemical and structural data indicate a potential homodimer. The proposed dimer model can be further positioned within the quaternary arrangement of the whole eEF1 assembly.     

Functional compatibility of elongation factors between mammalian mitochondrial and bacterial ribosomes: characterization of GTPase activity and translation elongation by hybrid ribosomes bearing heterologous L7/12 proteins

The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.     

eEF1A: Thinking Outside the Ribosome

Eukaryotic translation elongation factor 1A (eEF1A) is one of the most abundant protein synthesis factors. eEF1A is responsible for the delivery of all aminoacyl-tRNAs to the ribosome, aside from initiator and selenocysteine tRNAs. In addition to its roles in polypeptide chain elongation, unique cellular and viral activities have been attributed to eEF1A in eukaryotes from yeast to plants and mammals. From preliminary, speculative associations to well characterized biochemical and biological interactions, it is clear that eEF1A, of all the translation factors, has been ascribed the most functions outside of protein synthesis. A mechanistic understanding of these non-canonical functions of eEF1A will shed light on many important biological questions, including viral-host interaction, subcellular organization, and the integration of key cellular pathways.     

Interaction of the eukaryotic elongation factor 1A with newly synthesized polypeptides

eEF1A, the eukaryotic homologue of bacterial elongation factor Tu, is a well characterized translation elongation factor responsible for delivering aminoacyl-tRNAs to the A-site at the ribosome. Here we show for the first time that eEF1A also associates with the nascent chain distal to the peptidyltransferase center. This is demonstrated for a variety of nascent chains of different lengths and sequences. Interestingly, unlike other ribosome-associated factors, eEF1A also interacts with polypeptides after their release from the ribosome. We demonstrate that eEF1A does not bind to correctly folded full-length proteins but interacts specifically with proteins that are unable to fold correctly in a cytosolic environment. This association was demonstrated both by photo-cross-linking and by a functional refolding assay.