Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.     

Structure of yeast Dom34: a protein related to translation termination factor Erf1 and involved in No-Go decay

The yeast protein Dom34 has been described to play a critical role in a newly identified mRNA decay pathway called No-Go decay. This pathway clears cells from mRNAs inducing translational stalls through endonucleolytic cleavage. Dom34 is related to the translation termination factor eRF1 and physically interacts with Hbs1, which is itself related to eRF3. We have solved the 2.5-A resolution crystal structure of Saccharomyces cerevisiae Dom34. This protein is organized in three domains with the central and C-terminal domains structurally homologous to those from eRF1. The N-terminal domain of Dom34 is different from eRF1. It adopts a Sm-fold that is often involved in the recognition of mRNA stem loops or in the recruitment of mRNA degradation machinery. The comparison of eRF1 and Dom34 domains proposed to interact directly with eRF3 and Hbs1, respectively, highlights striking structural similarities with eRF1 motifs identified to be crucial for the binding to eRF3. In addition, as observed for eRF1 that enhances eRF3 binding to GTP, the interaction of Dom34 with Hbs1 results in an increase in the affinity constant of Hbs1 for GTP but not GDP. Taken together, these results emphasize that eukaryotic cells have evolved two structurally related complexes able to interact with ribosomes either paused at a stop codon or stalled in translation by the presence of a stable stem loop and to trigger ribosome release by catalyzing chemical bond hydrolysis.     

Crystal structures of two archaeal Pelotas reveal inter-domain structural plasticity

Dom34 from Saccharomyces cerevisiae is one of the key players in no-go mRNA decay, a surveillance pathway by which an abnormal mRNA stalled during translation is degraded by an endonucleolytic cleavage. Its homologs called Pelota are found in other species. We showed previously that S. cerevisiae Dom34 (domain 1) has an endoribonuclease activity, which suggests its direct catalytic role in no-go decay. Pelota from Thermoplasma acidophilum and Dom34 from S. cerevisiae have been structurally characterized, revealing a tripartite architecture with a significant difference in their overall conformations. To gain further insights into structural plasticity of the Pelota proteins, we have determined the crystal structures of two archaeal Pelotas from Archaeoglobus fulgidus and Sulfolobus solfataricus. Despite the structural similarity of their individual domains to those of T. acidophilum Pelota and S. cerevisiae Dom34, their overall conformations are distinct from those of T. acidophilum Pelota and S. cerevisiae Dom34. Different overall conformations are due to conformational flexibility of the two linker regions between domains 1 and 2 and between domains 2 and 3. The observed inter-domain structural plasticity of Pelota proteins suggests that large conformational changes are essential for their functions.     

Analysis of Dom34 and Its Function in No-Go Decay

Eukaryotic mRNAs are subject to quality control mechanisms that degrade defective mRNAs. In yeast, mRNAs with stalls in translation elongation are targeted for endonucleolytic cleavage by No-Go decay (NGD). The cleavage triggered by No-Go decay is dependent on Dom34p and Hbs1p, and Dom34 has been proposed to be the endonuclease responsible for mRNA cleavage. We created several Dom34 mutants and examined their effects on NGD in yeast. We identified mutations in several loops of the Dom34 structure that affect NGD. In contrast, mutations inactivating the proposed nuclease domain do not affect NGD in vivo. Moreover, we observed that overexpression of the Rps30a protein, a high copy suppressor of dom34Δ cold sensitivity, can restore some mRNA cleavage in a dom34Δ strain. These results identify important functional regions of Dom34 and suggest that the proposed endonuclease activity of Dom34 is not required for mRNA cleavage in NGD. We also provide evidence that the process of NGD is conserved in insect cells. On the basis of these results and the process of translation termination, we suggest a multistep model for the process of NGD.     

Structural and functional insights into Dom34, a key component of no-go mRNA decay

The yeast protein Dom34 is a key component of no-go decay, by which mRNAs with translational stalls are endonucleolytically cleaved and subsequently degraded. However, the identity of the endoribonuclease is unknown. Homologs of Dom34, called Pelota, are broadly conserved in eukaryotes and archaea. To gain insights into the structure and function of Dom34/Pelota, we have determined the structure of Pelota from Thermoplasma acidophilum (Ta Pelota) and investigated the ribonuclease activity of Dom34/Pelota. The structure of Ta Pelota is tripartite, and its domain 1 has the RNA-binding Sm fold. We have discovered that Ta Pelota has a ribonuclease activity and that its domain 1 is sufficient for the catalytic activity. We also demonstrate that domain 1 of Dom34 has an endoribonuclease activity against defined RNA substrates containing a stem loop, which supports a direct catalytic role of yeast Dom34 in no-go mRNA decay.     

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.     

Release Factor eRF3 Mediates Premature Translation Termination on Polylysine-Stalled Ribosomes in Saccharomyces cerevisiae

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.     

Evolution of nonstop,no-go and nonsense-mediated mRNA decay and their termination factor-derived components

Background  

Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis.     

Dom34‐Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress

Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation‐inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non‐translating 80S ribosomes stabilized by the ‘clamping’ Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34‐Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34‐Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34‐Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.     

Protein synthesis meets ABC ATPases: new roles for Rli1/ABCE1

The essential NTPase Rli1/ABCE1 has been implicated in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups —the former published in this issue of EMBO reports— suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes.EMBO Rep (2010) 11: 3 214–219. doi:10.1038/embor.2009.272The essential, conserved NTPase Rli1/ABCE1—a member of the ABC (ATP-binding cassette) superfamily of ATPases—has been implicated in translation initiation, ribosome biogenesis and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups—the former published in this issue of EMBO reports—suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).Two recent papers […] suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotesProtein synthesis is divided into four phases—initiation, elongation, termination and ribosome recycling—which are catalysed by several translation factors. The fundamental reactions of protein synthesis, such as mRNA decoding, peptide bond formation and tRNA translocation, follow the same basic principles in prokaryotes and eukaryotes. However, some steps are quite different and require a larger set of factors in eukaryotes. The best-studied example of eukaryotic complexity is the initiation of protein synthesis. In prokaryotes, initiation is catalysed by only three factors—IF1, IF2 and IF3—whereas in mammals it requires at least 13. Two recent papers shed new light on termination and ribosome recycling in the yeast and mammalian systems, suggesting that these two steps are also different in eukaryotes and prokaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).…new [research] on termination and ribosome recycling in the yeast and mammalian systems [suggests] that these two steps are also different in eukaryotes and prokaryotesIn prokaryotes, translation termination is promoted by three release factors: RF1, RF2 and RF3. RF1 and RF2 recognize the three stop codons and catalyse the hydrolysis of the peptidyl-tRNA. RF3, a GTP-binding protein that is not essential in bacteria, does not participate in peptide release but, at the expense of GTP hydrolysis, promotes the dissociation of RF1 and RF2, thereby accelerating their turnover (Kisselev et al, 2003). To free the ribosome for initiation on another mRNA (a process known as recycling), the post-termination ribosome is disassembled in a step that requires ribosome recycling factor (RRF) and one of the elongation factors, the GTPase EF-G. Together, these factors promote the dissociation of the post-termination complex into subunits. The subsequent dissociation of tRNA and mRNA from the small ribosomal subunit is promoted by initiation factors, in particular IF3 (Peske et al, 2005).In eukaryotes, translation termination is mediated by only two factors: eRF1 recognizes all three termination codons and triggers the hydrolysis of peptidyl-tRNA, whereas eRF3 accelerates the process in a GTP-dependent manner (Fig 1; Alkalaeva et al, 2006). Unlike prokaryotic RF1 or RF2—which have no measurable affinity for RF3—eRF1 binds tightly to eRF3, and it is probably the complex of the two proteins that enters the ribosome. The mechanism of guanine nucleotide exchange on eRF3 is also different from that on prokaryotic RF3, suggesting that termination in eukaryotes and prokaryotes differs in almost every detail except, probably, the mechanism of peptidyl-tRNA hydrolysis itself. Nevertheless, the identification of an additional factor that facilitates termination was unexpected. In this issue of EMBO reports, Khoshnevis and colleagues use the power of yeast genetics to show that a protein named Rli1 (RNase L inhibitor 1) interacts physically with the termination factors eRF1 (known as Sup45 in yeast) and, to a lesser extent, eRF3 (Sup35; Khoshnevis et al, 2010). The downregulation of Rli1 expression increases stop codon read-through in a dual reporter system, indicating a lower efficiency of termination. Conversely, upregulation of Rli1 partly suppresses the increased read-through caused by certain mutations of eRF1. Although the mechanism by which Rli1 affects translation termination is not understood, the results of the Krebber lab provide strong evidence that Rli1 mediates the function of eRF1 and eRF3 in vivo (Fig 1).…the identification [in eukaryotes] of an additional factor that facilitates termination was unexpectedOpen in a separate windowFigure 1New roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes. During termination, translating ribosomes contain peptidyl-tRNA (peptide is shown in dark blue and tRNA in dark red) in the P site and expose a stop codon in the A site. The stop codon is recognized by termination factor eRF1, which enters the ribosome together with eRF3-GTP. After GTP hydrolysis, catalysed by eRF3, the peptide is released from the peptidyl-tRNA with the help of eRF1. The point at which Rli1/ABCE1 binds to the ribosome is unknown, but the order shown is consistent with the effect of the factor on both termination and recycling. After NTP hydrolysis by Rli1/ABCE1, the 60S subunit and factors dissociate from the 40S subunit. Finally, tRNA and mRNA are released from the 40S subunit with the help of initiation factors (not shown). ABCE1, ATP-binding cassette, sub-family E member 1; eRF, eukaryotic release factor; Rli1, RNase L inhibitor 1.Surprisingly, modulating the efficiency of termination seems not to be the only function of Rli1 in translation. In a parallel study, Pestova and co-workers show that in higher eukaryotes, the homologue of Rli1—ABCE1—strongly enhances ribosome recycling (Pisarev et al, 2010). Eukaryotes lack a homologue of bacterial RRF and thus have to use other factors to disassemble the post-termination ribosome. Ribosome recycling can be brought about to some extent by eIF3, eIF1 and eIF1A (Pisarev et al, 2007), which is reminiscent of the IF3/IF1-mediated slow ribosome recycling that seems to occur in some conditions in bacterial systems. In eukaryotes, the initiation-factor-driven recycling operates only in a narrow range of low Mg²⁺ concentrations, probably because the affinity of the subunits to one another increases steeply with Mg²⁺ (Pisarev et al, 2010). By contrast, ABCE1 seems to catalyse efficient subunit dissociation in various conditions. To bind to the ribosome, ABCE1 requires the presence of eRF1, which is thought to induce a conformational change of the ribosome that unmasks the binding site for ABCE1. Subunit dissociation requires NTP (ATP, GTP, CTP or UTP) hydrolysis by ABCE1 (Fig 1). Subsequently, the dissociation of mRNA and tRNA from the small ribosomal subunit is promoted by initiation factors, which also inhibit the spontaneous reassociation of the subunits. Thus, the sequence of events during ribosome recycling in the eukaryotic system is remarkably similar to that in prokaryotes, and ABCE1 and eRF1 (possibly together with eRF3) seem to act as genuine ribosome recycling factors, similar to bacterial RRF/EF-G, despite the lack of any similarity in sequence or structure.Rli1/ABCE1 is a member of the ABC ATPases and comprises four structural domains (Karcher et al, 2008). Two nucleotide-binding domains (1 and 2) are connected by a hinge and arranged in a head-to-tail orientation. In contrast to other ABC enzymes, ABCE1 has an amino-terminal iron–sulphur (Fe–S) cluster domain, which is located in close proximity to, and presumably interacts with the nucleotide-binding loop of domain 1. Thus, there is a potential link between Fe–S domain function and NTP-induced conformational control of the ABC tandem cassette. Interestingly, although Khosnevis and colleagues map the eRF1 binding site on the second, carboxy-terminal ATPase domain, the Fe–S cluster is required for the function of Rli1/ABCE1 in termination and recycling (Khoshnevis et al, 2010). One might speculate that NTP hydrolysis is coupled to splitting the ribosome into subunits, in analogy to the prokaryotic recycling factors RRF/EF-G that couple the free energy of GTP hydrolysis and phosphate release into subunit dissociation (Savelsbergh et al, 2009). Kinetic experiments measuring single-round rates of subunit dissociation and NTP hydrolysis would be required to establish the existence and nature of such coupling.Another intriguing question is the role of the Fe–S cluster in Rli1/ABCE1. Fe–S protein biogenesis is the only known function of mitochondria that is indispensable for the viability of yeast cells (Lill, 2009). As yeast mitochondria do not contain essential Fe–S proteins, the essential character of the mitochondrial Fe–S protein assembly machinery could be attributed to its role in the maturation of extra-mitochondrial Fe–S proteins, such as Rli1/ABCE1, which is essential in all organisms tested.Another interesting finding by the Krebber group is that Rli1 can bind to Hcr1 (known as eIF3j in higher eukaryotes; Khoshnevis et al, 2010). Hcr1/eIF3j is an RNA-binding subunit of initiation factor eIF3, which is involved in initiation and required for Rli1/ABCE1-independent ribosome recycling. The fact that Rli1/ABCE1 binds to both eRF1 and Hcr1/eIF3j might indicate a functional or regulatory link between the termination, recycling and initiation machineries eukaryotes. It is unclear why eukaryotes require termination and recycling machinery that is so different from that of prokaryotes. One possibility is that Rli1/ABCE1, in contrast to its prokaryotic counterparts, not only acts in termination and recycling but also provides a platform for the recruitment of initiation factors to the ribosome, thereby acting as an additional checkpoint for translational control. Thus, the results of the Krebber and Pestova labs open a new, exciting avenue of research on eukaryotic protein synthesis.     

The nascent polypeptide in the 60S subunit determines the Rqc2-dependency of ribosomal quality control

Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.     

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.     

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

Structure of a human translation termination complex

In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the peptidyl-tRNA bond. While the molecular basis for bacterial termination has been elucidated, high-resolution structures of eukaryotic termination complexes have been lacking. Here we present a 3.8 Å structure of a human translation termination complex with eRF1 decoding a UAA(A) stop codon. The complex was formed using the human cytomegalovirus (hCMV) stalling peptide, which perturbs the peptidyltransferase center (PTC) to silence the hydrolysis activity of eRF1. Moreover, unlike sense codons or bacterial stop codons, the UAA stop codon adopts a U-turn-like conformation within a pocket formed by eRF1 and the ribosome. Inducing the U-turn conformation for stop codon recognition rationalizes how decoding by eRF1 includes monitoring geometry in order to discriminate against sense codons.     

Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.     

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.     

Receptor for activated C kinase 1 stimulates nascent polypeptide-dependent translation arrest

Nascent peptide-dependent translation arrest is crucial for the quality control of eukaryotic gene expression. Here we show that the receptor for activated C kinase 1 (RACK1) participates in nascent peptide-dependent translation arrest, and that its binding to the 40S subunit is crucial for this. Translation arrest by a nascent peptide results in Dom34/Hbs1-independent endonucleolytic cleavage of mRNA, and this is stimulated by RACK1. We propose that RACK1 stimulates the translation arrest that is induced by basic amino-acid sequences that leads to endonucleolytic cleavage of the mRNA, as well as to co-translational protein degradation.     

Eukaryotic Release Factor 3 Is Required for Multiple Turnovers of Peptide Release Catalysis by Eukaryotic Release Factor 1

Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.