The yeast translation release factors Mrf1p and Sup45p (eRF1) are methylated, respectively, by the methyltransferases Mtq1p and Mtq2p

The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N5-glutamine of the GGQ motif by PrmC methyltransferase. This motif is conserved in organisms from bacteria to higher eukaryotes. The Saccharomyces cerevisiae RFs, mitochondrial Mrf1p and cytoplasmic Sup45p (eRF1), have sequence similarities to the bacterial RFs, including the potential site of glutamine methylation in the GGQ motif. A computational analysis revealed two yeast proteins, Mtq1p and Mtq2p, that have strong sequence similarity to PrmC. Mass spectrometric analysis demonstrated that Mtq1p and Mtq2p methylate Mrf1p and Sup45p, respectively, in vivo. A tryptic peptide of Mrf1p, GGQHVNTTDSAVR, containing the GGQ motif was found to be approximately 50% methylated at the glutamine residue in the normal strain but completely unmodified in the peptide from mtq1-Delta. Moreover, Mtq1p methyltransferase activity was observed in an in vitro assay. In similar experiments, it was determined that Mtq2p methylates Sup45p. The Sup45p methylation by Mtq2p was recently confirmed independently (Heurgue-Hamard, V., Champ, S., Mora, L., Merkulova-Rainon, T., Kisselev, L. L., and Buckingham, R. H. (2005) J. Biol. Chem. 280, 2439-2445). Analysis of the deletion mutants showed that although mtq1-Delta had only moderate growth defects on nonfermentable carbon sources, the mtq2-Delta had multiple phenotypes, including cold sensitivity and sensitivity to translation fidelity antibiotics paromomycin and geneticin, to high salt and calcium concentrations, to polymyxin B, and to caffeine. Also, the mitochondrial mit(-) mutation, cox2-V25, containing a premature stop mutation, was suppressed by mtq1-Delta. Most interestingly, the mtq2-Delta was significantly more resistant to the anti-microtubule drugs thiabendazole and benomyl, suggesting that Mtq2p may also methylate certain microtubule-related proteins.     

The zinc finger protein Ynr046w is plurifunctional and a component of the eRF1 methyltransferase in yeast

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 A resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three alpha-helices. The active methyltransferase is the heterodimer Ydr140w.Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.     

Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo

Bacterial release factors RF1 and RF2 are methylated on the Gln residue of a universally conserved tripeptide motif GGQ, which interacts with the peptidyl transferase center of the large ribosomal subunit, triggering hydrolysis of the ester bond in peptidyl-tRNA and releasing the newly synthesized polypeptide from the ribosome. In vitro experiments have shown that the activity of RF2 is stimulated by Gln methylation. The viability of Escherichia coli K12 strains depends on the integrity of the release factor methyltransferase PrmC, because K12 strains are partially deficient in RF2 activity due to the presence of a Thr residue at position 246 instead of Ala. Here, we study in vivo RF1 and RF2 activity at termination codons in competition with programmed frameshifting and the effect of the Ala-246 --> Thr mutation. PrmC inactivation reduces the specific termination activity of RF1 and RF2(Ala-246) by approximately 3- to 4-fold. The mutation Ala-246 --> Thr in RF2 reduces the termination activity in cells approximately 5-fold. After correction for the decrease in level of RF2 due to the autocontrol of RF2 synthesis, the mutation Ala-246 --> Thr reduced RF2 termination activity by approximately 10-fold at UGA codons and UAA codons. PrmC inactivation had no effect on cell growth in rich media but reduced growth considerably on poor carbon sources. This suggests that the expression of some genes needed for optimal growth under such conditions can become growth limiting as a result of inefficient translation termination.     

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.     

Structures along the catalytic pathway of PrmC/HemK,an N5-glutamine AdoMet-dependent methyltransferase

Posttranslational methylation of release factors on the glutamine residue of a conserved GGQ motif is required for efficient termination of protein synthesis. This methylation is performed by an N(5)-glutamine methyltransferase called PrmC/HemK, whose crystal structure we report here at 2.2 A resolution. The electron density at the active site appears to contain a mixture of the substrates, S-adenosyl-L-methionine (AdoMet) and glutamine, and the products, S-adenosyl-L-homocysteine (AdoHcy) and N(5)-methylglutamine. The C-terminal domain of PrmC adopts the canonical AdoMet-dependent methyltransferase fold and shares structural similarity with the nucleotide N-methyltransferases in the active site, including use of a conserved (D/N)PPY motif to select and position the glutamine substrate. Residues of the PrmC (197)NPPY(200) motif form hydrogen bonds that position the planar Gln side chain such that the lone-pair electrons on the nitrogen nucleophile are oriented toward the methyl group of AdoMet. In the product complex, the methyl group remains pointing toward the sulfur, consistent with either an sp(3)-hybridized, positively charged Gln nitrogen, or a neutral sp(2)-hybridized nitrogen in a strained conformation. Due to steric overlap within the active site, proton loss and formation of the neutral planar methylamide product are likely to occur during or after product release. These structures, therefore, represent intermediates along the catalytic pathway of PrmC and show how the (D/N)PPY motif can be used to select a wide variety substrates.     

The essential role of the invariant GGQ motif in the function and stability in vivo of bacterial release factors RF1 and RF2

Release factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro, but these mutants could not complement thermosensitive RF mutants in vivo. None of several mutants with altered Gly residues retained activity in vivo or in vitro. Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.     

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.     

Molecular basis for bacterial class I release factor methylation by PrmC

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.     

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.     

Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis.

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.     

A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation

A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs. Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.     

The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors

Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.     

Release factors eRF1 and RF2: a universal mechanism controls the large conformational changes

Class I release factors 1 and 2 (RF1 and RF2) terminate protein synthesis by recognizing stop codons on the mRNA via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center. Crystal structures of eRF1 and RF2 do not fit their ribosomal binding pocket (approximately 73 angstroms). Cryoelectron microscopy indicates large conformational changes in the ribosome-bound RF2. Here, we investigate the conformational dynamics of the eRF1 and RF2 using molecular dynamics simulation, structural alignment, and electrostatic analysis of domain interactions. We show that relaxed eRF1 has a shape remarkably similar to the ribosome-bound RF2 observed by cryoelectron microscopy. The similarity between the two release factors is as good as between elongation factor G and elongation factor Tu-guanosine-5'(beta,gamma-imido)triphosphate-tRNA. Further, the conformational transitions and dynamics of eRF1 and RF2 between the free and ribosome-bound states are most likely controlled by protonation of conserved histidines. For eRF1, the distance between the NIKS and GGQ motifs shrinks from 97.5 angstroms in the crystal to 70-80 angstroms. For RF2, the separation between SPF and GGQ elongates from 32 angstroms in the crystal to 50 angstroms. Coulombic interaction strongly favors the open conformation of eRF1; however, solvation and histidine protonation modulate the domain interactions, making the closed conformation of eRF1 more accessible. Thus, RF1 and RF2 function like molecular machines, most likely fueled by histidine protonation. The unified conformational control and the shapes of eRF1 and RF2 support the proposition that the termination of protein synthesis involves similar mechanisms across species.     

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.     

Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1.

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.     

The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis

The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.     

The N5-glutamine S-adenosyl-L-methionine-dependent methyltransferase PrmC/HemK in Chlamydia trachomatis methylates class 1 release factors

The gene prmC, encoding the putative S-adenosyl-L-methionine (AdoMet)-dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis, was functionally analyzed. Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E. coli RFs in vivo. In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln. This is consistent with the enzymatic properties of PrmC of E. coli origin. We conclude that C. trachomatis PrmC functions as an N5-glutamine AdoMet-dependent MTase, involved in methylation of RFs.     

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.     

Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N⁶-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.Nucleic acids, proteins, carbohydrates, and lipids, as well as a body of small molecules, are subject to methylation in a wide variety of biological contexts (3). The majority of methylation reactions are catalyzed by S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). These enzymes ubiquitously exist in species from all three domains of life.Methylation of DNA occurs on one of two bases: cytosine or adenine (19). In prokaryotes, adenine methylation is as widespread as cytosine methylation. In contrast, eukaryotic genomes are devoid of adenine methylation or this type of methylation is too rare to be detected (23, 26). Intriguingly, two putative N⁶-adenine DNA MTases, named N6amt1 and N6amt2, are encoded in the mouse and human genomes. In addition to the conserved AdoMet-binding signature motif GXGXG and other sequence elements, they possess the NPPY motif characteristic of the N⁶-adenine or N⁴-cytosine DNA MTases in bacteria (6, 14). N6amt1 was thus proposed as an AdoMet-dependent DNA MTase, although no evidence had been provided that N6amt1 could methylate DNA (23).No functional clue for N6amt1 existed until two groups independently identified Escherichia coli HemK, distantly related to N6amt1, as a protein MTase for polypeptide release factors RF1 and RF2 (8, 17). The HemK gene was initially discovered in a genetic screen for heme biosynthesis mutants (18), although subsequent studies revealed no direct involvement in heme metabolism. The presence of an NPPY motif, thought to be restricted to members of the adenine and cytosine amino methyltransferases, led to the suggestion that HemK could be an AdoMet-dependent DNA MTase (2). However, a series of genetic and biochemical experiments finally revealed that HemK methylates the side-chain amide group of a glutamine residue in the universally conserved tripeptide motif GGQ of the two release factors in E. coli (8, 17). Methylation of the release factors ensures efficient translation termination and release of newly synthesized peptide from the ribosome (16). Similarly, the yeast HemK homologue, YDR140w (Mtq2p), was confirmed to methylate the eukaryotic release factor eRF1 on a corresponding glutamine residue (9, 22). More recently, the human homologue N6amt1 (HemK2) was reported to methylate release factor 1 (eRF1) in vitro (5).We initially sought to characterize the function of N6amt1 as a potential DNA adenine MTase. Interestingly, the human N6amt1 gene is located on chromosome 21q21.3, a critical region for Down syndrome (1, 20). In this study, we report the identification of murine N6amt1 as a glutamine-specific MTase of eRF1 both in vitro and in vivo. Mammalian eRF1, the only mammalian release factor, is indeed methylated at the glutamine residue of the GGQ motif. Inactivation of the N6amt1 gene by targeted disruption led to embryonic lethality in the mouse. These data confirm that N6amt1 functions as a protein MTase in mammals and indicate that modulation of the eRF1 activity by N6amt1-mediated glutamine methylation might be essential for embryo viability.     

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.