The Zinc Finger Protein Ynr046w Is Plurifunctional and a Component of the eRF1 Methyltransferase in Yeast

Abstract

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 Å 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 ␣-helices. The active methyltransferase is the heterodimer Ydr140w⅐Ynr046w, 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.Termination codons in mRNA are recognized on the ribosome by class I protein termination factors (or release factors (RFs)) 5 in eubacteria, archaea, and eukaryotes (1-3). Three codons are used as stop signals in most organisms: UAA, UGA, and UAG. In bacteria, two class I RFs are required for termination: RF1, which recognizes UAA and UAG codons, and RF2, which recognizes UAA and UGA codons. In contrast, a single RF, eRF1 or aRF1, is sufficient for termination at all three stop codons in eukaryotes and archaea, respectively. eRF1 and aRF1 form closely related protein families but are evolutionarily distinct from the eubacterial RFs (4). Thus, despite a similar function, the only sequence element common to all RFs is a tripeptide sequence, GGQ. Structural and mutational analysis shows that this motif is essential for RF activity and is required to interact with the peptidyl transferase center of the large ribosomal subunit and trigger hydrolysis of the ester bond in peptidyl-tRNA (5, 6). The Gln residue of the GGQ motif is methylated in both bacteria (7) and Saccharomyces cerevisiae (8, 9), and probably in mammals. Bacterial RF methylation depends on the PrmC methyltransferase (MTase) (10, 11), the product of the gene prmC (previously named hemK) situated in Escherichia coli, and most other bacteria immediately downstream of the gene prfA encoding RF1. RF methylation in E. coli strongly stimulates the activity of the factors (7, 11). 6 It is remarkable that the modification of Gln in the GGQ motif is conserved from bacteria to eukaryotes despite the different evolutionary origin of the class I RFs themselves. The S. cerevisiae genome encodes two proteins, Ydr140w and Ynl063w, with significant similarity to bacterial PrmC that goes beyond the motifs known to be involved in AdoMet binding (7). Inactivation of Ydr140w was shown to lead to a loss of eRF1 methylation ...

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

“…In that case, it was shown that co-expression of recombinant Trm112p with Mtq2p in Escherichia coli leads to the production of soluble Mtq2p that otherwise is recovered in the pellet of the bacterial extract. Co-expression of Trm112p with Mtq2p is also essential to reconstitute the catalytic activity of the recombinant protein (7). A similar conclusion was reached when Trm11p and Trm112p were produced together in vitro in a wheat germ cell-free extract (8).…”

“…Recently, it was reported that Trm11p alone, when produced in a wheat germ extract, had no tRNA methylation activity, whereas this activity was detected when Trm11p was synthesized together with Trm112p (8). Trm112p was also shown to be required for the activity of Mtq2p, the MTase of eRF1 (7). Therefore, Trm112p is essential for the activity of at least three different enzymes.…”

“…Trm112p co-precipitates with Trm11p and Mtq2q, and it improves the solubility of a recombinant Mtq2p when the two proteins are co-expressed in E. coli (7). To determine whether Trm112p could play a similar role on Trm9p, we prepared various constructs to express these two proteins in E. coli.…”

“…Trm11p is the catalytic subunit, whereas the function of Trm112p, although essential for the formation of m 2 G 10 , remains elusive. Trm112p is a small 15-kDa protein that contains a zinc finger domain (2,7). It is also required for the activity of Mtq2p, a protein methyltransferase that catalyzes the methylation of the glutamine of the universally conserved GGQ tripeptide of the translation termination factor eRF1/Sup45.…”

“…In one study, it was reported that the alteration of the translation release factor eRF1/Sup45 also leads to zymocin resistance (13). eRF1 possesses the universally conserved tripeptide GGQ, the Gln of which is methylated by the complex Mtq2-Trm112p (7,15,16). More specifically, a substitution of GGQ by the nonmethylatable sequences GGE or GGN induces strong zymocin resistance (13).…”