Mechanism of activation of methyltransferases involved in translation by the Trm112 'hub' protein

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.     

HemK2 protein, encoded on human chromosome 21, methylates translation termination factor eRF1

The ubiquitous tripeptide Gly-Gly-Gln in class 1 polypeptide release factors triggers polypeptide release on ribosomes. The Gln residue in both bacterial and yeast release factors is N5-methylated, despite their distinct evolutionary origin. Methylation of eRF1 in yeast is performed by the heterodimeric methyltransferase (MTase) Mtq2p/Trm112p, and requires eRF3 and GTP. Homologues of yeast Mtq2p and Trm112p are found in man, annotated as an N6-DNA-methyltransferase and of unknown function. Here we show that the human proteins methylate human and yeast eRF1.eRF3.GTP in vitro, and that the MTase catalytic subunit can complement the growth defect of yeast strains deleted for mtq2.     

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.     

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.     

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.     

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.     

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.     

Translation termination and its regulation in eukaryotes: recent insights provided by studies in yeast

In protein synthesis, the arrival of one or other of the three stop codons in the ribosomal A-site triggers the binding of a release factor (RF) to the ribosome and subsequent polypeptide chain release. In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3. eRF1 is responsible for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function, although its precise role remains to be defined. Recent findings on translation termination and its regulation from studies in the yeast Saccharomyces cerevisiae are reviewed and the potential role of eRF3 is discussed.     

Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.     

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.     

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.     

The iron–sulphur protein RNase L inhibitor functions in translation termination

The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.     

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.     

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.     

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.     

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm⁵U₃₄ and mcm⁵s²U₃₄), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm⁵U₃₄ and mcm⁵s²U₃₄. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm⁵U₃₄ on tRNA in vitro but not mcm⁵s²U₃₄. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.