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 RFs ensures efficient translation termination and release of newly synthesized peptides from the ribosome [19, 22]. Methylation of eRF1 in yeast is performed by the heterodimeric methyltransferase (MTase) Mtq2/Trm112 at the glutamine residue of the GGQ motift (ripeptide Gly-Gly-Gln) [4, 22], and Trm112 is necessary for the solubility and activity of the catalytic subunit of the protein methylase Mtq2p [5, 26]. More recently, the same mechanism was also found in human and mouse, the holoenzyme homologues of which are annotated as HEMK2α/hTRM112 and Pred28a (N6amt1)/mTrm112, respectively [2, 13, 14, 17, 24]. "
[Show abstract][Hide abstract] ABSTRACT: Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.
"In both bacteria and eukarya, this motif is post-translationally modified by methylation, one of the most common modifications after phosphorylation. The Gln side chain of this motif is converted into N 5 methyl-glutamine by specific protein methyltransferases (MTases): PrmC in Escherichia coli or the Mtq2-Trm112 complex in Saccharomyces cerevisiae  . "
[Show abstract][Hide abstract] ABSTRACT: During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies.
"First, Trm112 masks a hydrophobic region from Mtq2 upon complex formation, explaining the need for co-expression of Trm112 and Mtq2 to solubilize Mtq2 (Supplementary Figure 3D and E) (22). The same mechanism may be shared by Trm9, which can only be recovered as a soluble protein upon co-expression with Trm112 (22,27) (M.G. and V.H.H., unpublished data). Second, Trm112 stimulates SAM-binding by Mtq2 via its interaction with the Mtq2 loop connecting strands β3–β4, which in turn contacts the SAM molecule (Figure 1B and C). "
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 04/2011; 39(14):6249-59. DOI:10.1093/nar/gkr176 · 9.11 Impact Factor
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