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J. Biol. Chem., Vol. 282, Issue 38, 27744-27753, September 21, 2007

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Yeast Mitochondrial Initiator tRNA Is Methylated at Guanosine 37 by the Trm5-encoded tRNA (Guanine-N1-)-methyltransferase^*

From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m¹G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m¹G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA^Met_f). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA^Met_f and tRNA^Phe. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1–33 (1–33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the 1–33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m¹G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.

Received for publication, June 4, 2007 , and in revised form, July 5, 2007.

^* This work was supported in part by Robert A. Welch Foundation Grant F-1576 (to D. E. G.) and the U. S. Army Research Office Grant W911NF-07-1-0040 (to D. R. A. and G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Tables S1 and S2, and Figs. S1–S3.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin TX 78712-0165. Tel.: 512-471-5842; Fax: 512-471-5849; E-mail: dappling{at}mail.utexas.edu.

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