Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons

In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.     

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.     

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.     

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.     

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.     

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.     

Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast

Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.     

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。     

Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity

Class 1 release factor in eukaryotes (eRF1) recognizes stop codons and promotes peptide release from the ribosome. The ‘molecular mimicry’ hypothesis suggests that domain 1 of eRF1 is analogous to the tRNA anticodon stem–loop. Recent studies strongly support this hypothesis and several models for specific interactions between stop codons and residues in domain 1 have been proposed. In this study we have sequenced and identified novel eRF1 sequences across a wide diversity of eukaryotes and re-evaluated the codon-binding site by bioinformatic analyses of a large eRF1 dataset. Analyses of the eRF1 structure combined with estimates of evolutionary rates at amino acid sites allow us to define the residues that are under structural (i.e. those involved in intramolecular interactions) versus non-structural selective constraints. Furthermore, we have re-assessed convergent substitutions in the ciliate variant code eRF1s using maximum likelihood-based phylogenetic approaches. Our results favor the model proposed by Bertram et al. that stop codons bind to three ‘cavities’ on the protein surface, although we suggest that the stop codon may bind in the opposite orientation to the original model. We assess the feasibility of this alternative binding orientation with a triplet stop codon and the eRF1 domain 1 structures using molecular modeling techniques.     

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.     

New insights into stop codon recognition by eRF1

In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.     

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.     

A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons.     

Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition

Eukaryote ribosomal translation is terminated when release factor eRF1, in a complex with eRF3, binds to one of the three stop codons. The tertiary structure and dimensions of eRF1 are similar to that of a tRNA, supporting the hypothesis that release factors may act as molecular mimics of tRNAs. To identify the yeast eRF1 stop codon recognition domain (analogous to a tRNA anticodon), a genetic screen was performed to select for mutants with disabled recognition of only one of the three stop codons. Nine out of ten mutations isolated map to conserved residues within the eRF1 N-terminal domain 1. A subset of these mutants, although wild-type for ribosome and eRF3 interaction, differ in their respective abilities to recognize each of the three stop codons, indicating codon-specific discrimination defects. Five of six of these stop codon-specific mutants define yeast domain 1 residues (I32, M48, V68, L123, and H129) that locate at three pockets on the eRF1 domain 1 molecular surface into which a stop codon can be modeled. The genetic screen results and the mutant phenotypes are therefore consistent with a role for domain 1 in stop codon recognition; the topology of this eRF1 domain, together with eRF1-stop codon complex modeling further supports the proposal that this domain may represent the site of stop codon binding itself.     

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.     

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.     

Identification of eRF1 residues that play critical and complementary roles in stop codon recognition

The initiation and elongation stages of translation are directed by codon-anticodon interactions. In contrast, a release factor protein mediates stop codon recognition prior to polypeptide chain release. Previous studies have identified specific regions of eukaryotic release factor one (eRF1) that are important for decoding each stop codon. The cavity model for eukaryotic stop codon recognition suggests that three binding pockets/cavities located on the surface of eRF1's domain one are key elements in stop codon recognition. Thus, the model predicts that amino acid changes in or near these cavities should influence termination in a stop codon-dependent manner. Previous studies have suggested that the TASNIKS and YCF motifs within eRF1 domain one play important roles in stop codon recognition. These motifs are highly conserved in standard code organisms that use UAA, UAG, and UGA as stop codons, but are more divergent in variant code organisms that have reassigned a subset of stop codons to sense codons. In the current study, we separately introduced TASNIKS and YCF motifs from six variant code organisms into eRF1 of Saccharomyces cerevisiae to determine their effect on stop codon recognition in vivo. We also examined the consequences of additional changes at residues located between the TASNIKS and YCF motifs. Overall, our results indicate that changes near cavities two and three frequently mediated significant effects on stop codon selectivity. In particular, changes in the YCF motif, rather than the TASNIKS motif, correlated most consistently with variant code stop codon selectivity.     

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.     

Decoding the Decoding Region: Analysis of Eukaryotic Release Factor (eRF1) Stop Codon-Binding Residues

Peptide synthesis in eukaryotes terminates when eukaryotic release factor 1 (eRF1) binds to an mRNA stop codon and occupies the ribosomal A site. Domain 1 of the eRF1 protein has been implicated in stop codon recognition in a number of experimental studies. In order to further pinpoint the residues of this protein involved in stop codon recognition, we sequenced and compared eRF1 genes from a variety of ciliated protozoan species. We then performed a series of computational analyses to evaluate the conservation, accessibility, and structural environment of each amino acid located in domain 1. With this new dataset and methodology, we were able to identify eight specific amino acid sites important for stop codon recognition and also to propose a set of cooperative paired substitutions that may underlie stop codon reassignment. Our results are more consistent with current experimental data than previously described models.Han Liang, Jonathan Y. Wong,Contributed equally to this paperReviewingEditor: Dr. Niles Lehman     

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.