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.     

Identification of eRF3b,a Human Polypeptide Chain Release Factor with eRF3 Activity in vitroand 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 vitroas a potent stimulator of the release factor activity of human eRFl. Like eRF3a, eRF3b exhibits GTPase activity, which is ribosome- and eRFl-dependent. In vivoassays (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 eRF1 with all three stop codons.     

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

In eukaryotes, eRF1 and eRF3 are associated in a complex that mediates translation termination. The regulation of the formation of this complex in vivo is far from being understood. In mammalian cells, depletion of eRF3a causes a reduction of eRF1 level by decreasing its stability. Here, we investigate the status of eRF3a when not associated with eRF1. We show that eRF3a forms altered in their eRF1-binding site have a decreased stability, which increases upon cell treatment with the proteasome inhibitor MG132. We also show that eRF3a forms altered in eRF1 binding as well as wild-type eRF3a are polyubiquitinated. These results indicate that eRF3a is degraded by the proteasome when not associated with eRF1 and suggest that proteasomal degradation of eRF3a controls translation termination complex formation by adjusting the eRF3a level to that of eRF1.     

Increased tRNA level in yeast cells with mutant translation termination factors eRF1 and eRF3

We have earlier characterized Saccharomyces cerevisiae strains with mutations of essential SUP45 and SUP35, which code for translation termination factors eRF1 and eRF3, respectively. In this work, the sup45 and sup35 nonsense mutants were compared with respect to the levels of eight tRNAs: tRNA^Tyr, tRNA^Gln, tRNA^Trp, tRNA^Leu, tRNA^Arg (described as potential suppressor tRNAs), tRNA^Pro, tRNA^His, and tRNA^Gly. The mutants did not display a selective increase in tRNAs, capable of a noncanonical read-through at stop codons. Most of the mutations increased the level of all tRNAs under study. The mechanisms providing for the viability of the sup45 and sup35 nonsense mutants are discussed.     

RF3:GTP promotes rapid dissociation of the class 1 termination factor

Translation termination is promoted by class 1 and class 2 release factors in all domains of life. While the role of the bacterial class 1 factors, RF1 and RF2, in translation termination is well understood, the precise contribution of the bacterial class 2 release factor, RF3, to this process remains less clear. Here, we use a combination of binding assays and pre-steady state kinetics to provide a kinetic and thermodynamic framework for understanding the role of the translational GTPase RF3 in bacterial translation termination. First, we find that GDP and GTP have similar affinities for RF3 and that, on average, the t_1/2 for nucleotide dissociation from the protein is 1–2 min. We further show that RF3:GDPNP, but not RF3:GDP, tightly associates with the ribosome pre- and post-termination complexes. Finally, we use stopped-flow fluorescence to demonstrate that RF3:GTP enhances RF1 dissociation rates by over 500-fold, providing the first direct observation of this step. Importantly, catalytically inactive variants of RF1 are not rapidly dissociated from the ribosome by RF3:GTP, arguing that a rotated state of the ribosome must be sampled for this step to efficiently occur. Together, these data define a more precise role for RF3 in translation termination and provide insights into the function of this family of translational GTPases.     

Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome

To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31–33, and to lesser extent amino acids within region 121–131 (the “YxCxxxF loop”) in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61–64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of “protein anticodon” type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31–33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.     

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.     

A New Method to Measure the Functional Activity of Class-1 Translation Termination Factor eRF1

Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.     

NMR assignments of the C-terminal domain of human polypeptide release factor eRF1

We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states.     

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.     

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.     

Molecular Morphology of Eukaryotic Class-1 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 100–107 Å). 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.     

Stop codons and UGG promote efficient binding of the polypeptide release factor eRF1 to the ribosomal A site

To investigate the codon dependence of human eRF1 binding to the mRNA-ribosome complex, we examined the formation of photocrosslinks between ribosomal components and mRNAs bearing a photoactivable 4-thiouridine probe in the first position of the codon located in the A site. Addition of eRF1 to the phased mRNA-ribosome complexes triggers a codon-dependent quenching of crosslink formation. The concentration of eRF1 triggering half quenching ranges from low for the three stop codons, to intermediate for s4UGG and high for other near-cognate triplets. A theoretical analysis of the photochemical processes occurring in a two-state bimolecular model raises a number of stringent conditions, fulfilled by the system studied here, and shows that in any case sound KD values can be extracted if the ratio mT/KD<1 (mT is total concentration of mRNA added). Considering the KD values obtained for the stop, s4UGG and sense codons (approximately 0.06 microM, 0.45 microM and 2.3 microM, respectively) and our previous finding that only the stop and s4UGG codons are able to promote formation of an eRF1-mRNA crosslink, implying a role for the NIKS loop at the tip of the N domain, we propose a two-step model for eRF1 binding to the A site: a codon-independent bimolecular step is followed by an isomerisation step observed solely with stop and s4UGG codons. Full recognition of the stop codons by the N domain of eRF1 triggers a rearrangement of bound eRF1 from an open to a closed conformation, allowing the universally conserved GGQ loop at the tip of the M domain to come into close proximity of the peptidyl transferase center of the ribosome. UGG is expected to behave as a cryptic stop codon, which, owing to imperfect eRF1-codon recognition, does not allow full reorientation of the M domain of eRF1. As far as the physical steps of eRF1 binding to the ribosome are considered, they appear to closely mimic the behaviour of the tRNA/EF-Tu/GTP complex, but clearly eRF1 is endowed with a greater conformational flexibility than tRNA.     

Mutation at tyrosine in AMLRY (GILRY like) motif of yeast eRF1 on nonsense codons suppression and binding affinity to eRF3

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain.     

N-terminal extension of Saccharomyces cerevisiae translation termination factor eRF3 influences the suppression efficiency of sup35 mutations

The eukaryotic translation termination factor eRF3 stimulates release of nascent polypeptides from the ribosome in a GTP-dependent manner. In most eukaryotes studied, eRF3 consists of an essential, conserved C-terminal domain and a nonessential, nonconserved N-terminal extension. However, in some species, this extension is required for efficient termination. Our data show that the N-terminal extension of Saccharomyces cerevisiae eRF3 also participates in regulation of termination efficiency, but acts as a negative factor, increasing nonsense suppression efficiency in sup35 mutants containing amino acid substitutions in the C-terminal domain of the protein.     

Structure and dynamics in solution of the stop codon decoding N-terminal domain of the human polypeptide chain release factor eRF1

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.     

Cloning, characterization and expression of the polypeptide release factor gene, eRF1, of Blepharisma japonicum

The polypeptide release factor gene, eRF1, of Blepharisma japonicum (Bj-eRF1) was cloned and sequenced. Its coding region was 1314 base pairs and encodes a protein of 437 amino acids. The cloned gene was expressed in Escherichia coli and the recombinant Bj-eRF1 polypeptide was purified by Ni2+-nitrilotriacetic acid agarose and Superose12 chromatography. Pull-down analysis showed that the recombinant Bj-eRF1 interacts with the heterologously-expressed release factor, eRF3C, of Euplotes octocarinatus.     

第一类肽链释放因子C端结构域参与调控终止密码子的识别过程

真核生物蛋白质翻译终止过程中,第一类肽链释放因子（eukaryotic polypeptide release factor, eRF1）利用其N端结构域识别终止密码子。eRF1的N结构域中的GTS、NIKS和YxCxxxF模体对于终止密码子的识别发挥重要作用。但至目前为止,eRF1识别终止密码子的机制,尤其是对于终止密码子的选择性识别机制仍不清楚。我们构建了四膜虫（Tetrahymena thermophilia）eRF1的N端结构域与酿酒酵母（Saccharomyces cerevisiae）或裂殖酵母（Schizosaccharomyces pombe）eRF1的M和C结构域组成的杂合eRF1,即Tt/Sc eRF1 和Tt/Sp eRF1。双荧光素酶检测结果证实,两种杂合eRF1在细胞中识别终止密码子的活性具有显著差异。Tt/Sc eRF1仅识别UGA密码子,与四膜虫eRF1一致,具有密码子识别特异性;而Tt/Sp eRF1可以识别3个终止密码子,无密码子识别特异性。为解释这一现象,将Sp eRF1的C结构域中的1个关键的小结构域中的氨基酸进行突变,与Sc eRF1相应位点的氨基酸一致。分析结果显示,突变体Tt/Sp eRF1识别密码子UAA和UAG的性质发生显著变化,说明第一类肽链释放因子的C端结构域参与了终止密码子的识别过程。这提示,四膜虫eRF1识别终止密码子的特异性可能依赖于eRF1分子内的结构域间相互作用。本研究结果为揭示肽链释放因子识别终止密码子的分子机制提供了数据支持。     

Class-1 polypeptide chain release factors are structurally and functionally similar to suppressor tRNAs and comprise different structural-functional families of prokaryotic/mitochondrial and eukaryotic/archaebacterial factors

Class-1 polypeptide chain release factors (RF) induce peptidyl-tRNA hydrolysis in the ribosome if any of the three stop codons encounters the ribosomal A site. We have shown earlier that all factors of this class possess a common functionally essential motif GGQ. In this study we analyzed the primary structures of all known class-1 factors taken from the data banks together with the experimental data available on their structural and functional organization. The following conclusions were drawn. 1. Amino acid sequences of eukaryotic and archaebacterial factors (eRF1 and aRF1, respectively) show high similarity. This suggests the potential ability of eRF1 to function in archaebacterial and aRF1 in eukaryotic ribosomes, and points to their origin from a common ancestor. 2. Primary structures of class-1 release factors from prokaryotes and enkaryotic mitochondria show no statistically significant similarity with archaebacterial and cytoplasmic eukaryotic release factors, except for a common motif GGQ. This confirms our earlier conclusion (Nature, 1994, vol. 372, pp. 701–703) and contradicts the hypothesis of Itoet al. (Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 5443–5448) about structural similarity of all class-1 release factors. 3. All the eRF1/aRF1 recognizing three stop codons have a common motif NIKs that is absent from eubacterial RF1 and RF2, each of which is able to recognize two stop codons of the three. We suppose that the function of the NIKs motif is to fix the proper orientation of eRF1/aRF1 at the ribosome. 4. The domain structure and functional properties of eRF1/aRF1 point to the similarity of these factors with suppressor tRNAs as suggested long ago, and also semblance with aminoacyl-tRNA synthetases. 5. Considering that peptidyl-tRNA is fixed at the ribosomal P site while the stop codon and termination factor are at the A site, it may be presumed that the distance between the functionally essential motifs NIKs and GGQS in eRF1/aRF1 should approximately correspond to the distance between the anticodon and the aminoacyl end of aminoacyl-tRNA located at the ribosomal A site.