Conservation of the MC domains in eukaryotic release factor eRF3

Eukaryotic translation termination employs two protein factors, eRF1 and eRF3. Proteins of the eRF3 family each consist of three domains. The N and M domains vary in different species, while the C domains are highly homologous. The MC domains of Homo sapiens eRF3a (hGSPT1), Xenopus laevis eRF3 (XSup35), and Mus musculus eRF3a (mGSPT1) and eRF3b (mGSPT2) were found to compensate for the sup35-21(ts) temperature-sensitive mutation and lethal disruption of the SUP35 gene in yeast Saccharomyces cerevisiae. At the same time, strains containing the MC domains of the eRF3 proteins from different species differed in growth rate and the efficiency of translation termination.     

Role of the individual domains of translation termination factor eRF1 in GTP binding to eRF3

Eukaryotic translational termination is triggered by polypeptide release factors eRF1, eRF3, and one of the three stop codons at the ribosomal A-site. Isothermal titration calorimetry shows that (i) the separated MC, M, and C domains of human eRF1 bind to eRF3; (ii) GTP binding to eRF3 requires complex formation with either the MC or M + C domains; (iii) the M domain interacts with the N and C domains; (iv) the MC domain and Mg2+ induce GTPase activity of eRF3 in the ribosome. We suggest that GDP binding site of eRF3 acquires an ability to bind gamma-phosphate of GTP if altered by cooperative action of the M and C domains of eRF1. Thus, the stop-codon decoding is associated with the N domain of eRF1 while the GTPase activity of eRF3 is controlled by the MC domain of eRF1 demonstrating a substantial structural uncoupling of these two activities though functionally they are interrelated.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins belonging to class 1 (eRF1) and class 2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to yield GDP and P_i in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA, and peptidyl-tRNA and requires eRF1 for this activity. It is known that eRF1 and eRF3 interact with each other via their C-terminal regions both in vitro and in vivo. eRF1 consists of three domains—N, M, and C. In this study we examined the influence of the individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N, M, C, and NM domains induces the eRF3 GTPase activity in the presence of ribosomes. The MC domain does induce the eRF3 GTPase activity, but four times less efficiently than full-length eRF1. Therefore, we assumed that the MC domain (and very likely the M domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in the literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated that the eRF3 GTPase activity in the ribosome during translation termination is associated with the intermolecular interactions of GTP/GDP, the GTPase center of the large (60S) subunit, the MC domain of eRF1, and the C-terminal region and GTP-binding motifs of eRF3 but without participation of the N-terminal region of eRF1.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.     

Molecular morphology of eukaryotic class I translation termination factor eRF1 in solution

The integral structural parameters and the shape of the molecule of human translation termination factor eRF1 were determined from the small-angle X-ray scattering in solution. The molecular shapes were found by bead modeling with nonlinear minimization of the root-mean-square deviation of the calculated from the experimental scattering curve. Comparisons of the small-angle scattering curves computed for atomic-resolution structures of eRF1 with the experimental data on scattering from solution testified that the crystal and the solution conformations are close. In the ribosome, the distance between the eRF1 motifs GGQ and NIKS must be shorter than in crystal or solution (75 versus 107-112 A). Therefore, like its bacterial counterpart RF2, the eukaryotic eRF1 must change its conformation as it binds to the ribosome. The conformational mobility of eukaryotic and prokaryotic class-1 release factors is another feature making them functionally akin to tRNA.     

Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.     

Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1

Class-1 polypeptide chain release factors (RFs) play a key role in translation termination. Eukaryotic (eRF1) and archaeal class-1 RFs possess a highly conserved Asn-Ile-Lys-Ser (NIKS) tetrapeptide located at the N-terminal domain of human eRF1. In the three-dimensional structure, NIKS forms a loop between helices. The universal occurrence and exposed nature of this motif provoke the appearance of hypotheses postulating an essential role of this tetrapeptide in stop codon recognition and ribosome binding. To approach this problem experimentally, site-directed mutagenesis of the NIKS (positions 61-64) in human eRF1 and adjacent amino acids has been applied followed by determination of release activity and ribosome-binding capacity of mutants. Substitutions of Asn61 and Ile62 residues of the NIKS cause a decrease in the ability of eRF1 mutants to promote termination reaction in vitro, but to a different extent depending on the stop codon specificity, position, and nature of the substituting residues. This observation points to a possibility that Asn-Ile dipeptide modulates the specific recognition of the stop codons by eRF1. Some replacements at positions 60, 63, and 64 cause a negligible (if any) effect in contrast to what has been deduced from some current hypotheses predicting the structure of the termination codon recognition site in eRF1. Reduction in ribosome binding revealed for Ile62, Ser64, Arg65, and Arg68 mutants argues in favor of the essential role played by the right part of the NIKS loop in interaction with the ribosome, most probably with ribosomal RNA.     

Identification of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and in vivo

Termination of translation in eukaryotes is governed by the ribosome, a termination codon in the mRNA, and two polypeptide chain release factors (eRF1 and eRF3). We have identified a human protein of 628 amino acids, named eRF3b, which is highly homologous to the known human eRF3 henceforth named eRF3a. At the nucleotide and at the amino acid levels the human eRF3a and eRF3b are about 87% identical. The differences in amino acid sequence are concentrated near the amino terminus. The most important difference in the nucleotide sequence is that eRF3b lacks a GGC repeat close to the initiation codon in eRF3a. We have cloned the cDNA encoding the human eRF3b, purified the eRF3b expressed in Escherichia coli, and found that the protein is active in vitro as a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivo assays (based on suppression of readthrough induced by three species of suppressor tRNAs: amber, ochre, and opal) show that the human eRF3b is able to enhance the release factor activity of endogenous and overexpressed eRFl with all three stop codons.     

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.     

Monoclonal antibodies against human translation termination factor eRF3 and their utilization for sub-cellular localization of eRF3

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes the stop codon and promotes nascent peptide chain release, while eRF3 facilitates this peptide release in a GTP-dependent manner. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay. Despite extensive investigation, the complete understanding of eRF3 function have been hampered by the lack of specific anti-eRF3 monoclonal antibodies (Mabs). The purpose of the study was production of recombinant eRF3a/GSPT1, development of anti-eRF3a/GSPT1 Mabs and their utilization for eRF3a/GSPT1 sub-cellular localization. Plasmid encoding C-terminal part of human GSPT1/eRF3a was constructed. Purified protein, which was predominantly present in the inclusion bodies, was used for the development of Mabs. Characterization of the regions recognized by Mabs using GSPT1/eRF3a mutants and its visualization in the 3D space suggested that Mabs recognize different epitopes. Consistent with its function in translational termination, immunostaining of the cells with developed Mabs revealed that the endogenous GSPT1/eRF3a localized in endoplasmic reticulum. Taking into account the important role of eRF3 for the fundamental research one can suggests that developed Mabs have great prospective to be used as a research reagent in a wide range of applications.     

Interface of the interaction of the middle domain of human translation termination factor eRF1 with eukaryotic ribosomes

Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix α1 of the M domain. Some residues adjacent to α1 and belonging to strand β5 and short helices α2 and α3 are also involved in the protein-ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix α1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight protein-ribosome contact.     

Thermal denaturation of class 1 eukaryotic translation termination factor eRF1. Relationship between stability and functional activity of eRF1 mutants

Differential scanning calorimetry (DSC) was used to study thermal denaturation of the human class 1 translation termination factor eRF1 and its mutants. Free energy changes caused by amino acid substitutions in the N domain were computed for eRF1. The melting of eRF1, consisting of three domains, proved to be cooperative. The thermostability of eRF1 was not affected by certain substitutions and was slightly increased by certain others. The corresponding residues were assumed to play no role in maintaining the eRF1 structure, which agreed with the published X-ray data. In these mutants (E55D, Y125F, N61S, E55R, E55A, N61S + S64D, C127A, and S64D), a selective loss of the capability to induce hydrolysis of peptidyl-tRNA in the ribosomal P site in the presence of a stop codon was not associated with destabilization of their spatial structure. Rather, the loss was due to local changes in the stereochemistry of the side groups of the corresponding residues in functionally important sites of the N domain. Two amino acid residues of the N domain, N129 and F131, proved to play an important role in the structural stability of eRF1 and to affect the selective recognition of mRNA stop codons in the ribosome. The recognition of the UAG and UAA stop codons in vitro was more tightly associated with the stability of the spatial structure of eRF1 as compared with that of the UGA stop codon.     

Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation

Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end. However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated. Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae. The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP. When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged. The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening. Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon. These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.     

Involvement of human release factors eRF3a and eRF3b in translation termination and regulation of the termination complex formation

eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.     

Conversion of omnipotent translation termination factor eRF1 into ciliate-like UGA-only unipotent eRF1

In eukaryotic ribosomes, termination of translation is triggered by class 1 polypeptide release factor, eRF1. In organisms with a universal code, eRF1 responds to three stop codons, whereas, in ciliates with variant codes, only one or two codon(s) remain(s) as stop signals. By mutagenesis of the Y-C-F minidomain of the N domain, we converted an omnipotent human eRF1 recognizing all three stop codons into a unipotent 'ciliate-like' UGA-only eRF1. The conserved Cys127 located in the Y-C-F minidomain plays a critical role in stop codon recognition. The UGA-only response has also been achieved by concomitant substitutions of four other amino acids located at the Y-C-F and NIKS minidomains of eRF1. We suggest that for eRF1 the stop codon decoding is of a non-linear (non-protein-anticodon) type and explores a combination of positive and negative determinants. We assume that stop codon recognition is profoundly different by eukaryotic and prokaryotic class 1 RFs.     

Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.     

Evaluation of the gene encoding translation termination factor eRF3 as a possible phylogenetic marker

The region of the nuclear GSPT2 gene coding for the N and M domains of translation termination factor eRF3b was tested in Rodentia for applicability as a new molecular marker. It cannot be used as a phylogenetic marker at the intrageneric level because of insufficient variability within families and the impossibility of resolving relationships in the family Cricetidae. However, this GSPT2 region allows reliable identification of higher taxa. The phylogenetic relationships among families revealed with the proposed molecular marker is generally in agreement with current concepts. The new marker indicates a close relationship between the genus Acomys and the family Gerbillidae, which is in agreement with other molecular data but contradicts morphological data. Thus, the region of the nuclear GSPT2 gene encoding the N and M domains of eRF3b can serve as an adequate phylogenetic marker in placental mammals at the level of families or higher taxa. It can also be used in solving controversial questions of phylogeny and taxonomy.     

Nuclear localization of eukaryotic class II release factor (eRF3): implication for the multifunction of eRF3 in ciliates Euplotes cell

Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis.     

N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination

Background  

Termination of translation in eukaryotes requires two release factors, eRF1, which recognizes all three nonsense codons and facilitates release of the nascent polypeptide chain, and eRF3 stimulating translation termination in a GTP-depended manner. eRF3 from different organisms possess a highly conservative C region (eRF3C), which is responsible for the function in translation termination, and almost always contain the N-terminal extension, which is inessential and vary both in structure and length. In the yeast Saccharomyces cerevisiae the N-terminal region of eRF3 is responsible for conversion of this protein into the aggregated and functionally inactive prion form.     

Elongation factor eEF1B modulates functions of the release factors eRF1 and eRF3 and the efficiency of translation termination in yeast

Background  

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation.