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J. Biol. Chem., Vol. 282, Issue 32, 23663-23671, August 10, 2007

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The Carboxyl-terminal Extension of Yeast tRNA m⁵C Methyltransferase Enhances the Catalytic Efficiency of the Amino-terminal Domain^*

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS Bâtiment 34, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France

The human tRNA m⁵C methyltransferase is a potential target for anticancer drugs because it is a novel downstream target of the proto-oncogene myc, mediating Myc-induced cell proliferation. Sequence comparisons of RNA m⁵C methyltransferases indicate that the eukaryotic enzymes possess, in addition to a conserved catalytic domain, a large characteristic carboxyl-terminal extension. To gain insight into the function of this additional domain, the modular architecture of the yeast tRNA m⁵C methyltransferase orthologue, Trm4p, was studied. The yeast enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosine at different positions depending on the tRNAs. By limited proteolysis, Trm4p was shown to be composed of two domains that have been separately produced and purified. Here we demonstrate that the aminoterminal domain, encompassing the active site, binds tRNA with similar affinity as the whole enzyme but shows low catalytic efficiency. The carboxyl-terminal domain displays only weak affinity for tRNA. It is not required for m⁵C formation and does not appear to contribute to substrate specificity. However, it enhances considerably the catalytic efficiency of the amino-terminal domain.

Received for publication, May 9, 2007 , and in revised form, June 13, 2007.

^* 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 Fig. S1.

¹ Both authors contributed equally to this work.

² Supported by a fellowship from the Association pour la Recherche sur le Cancer.

³ Present address: Université Paris-Sud-11, Institut de Génétique et Microbiologie, Bâtiment 400, 91400 Orsay, France.

⁴ To whom correspondence should be addressed. Tel.: 33-1-69-82-42-35; Fax: 33-1-69-82-31-29; E-mail: beatrice.golinelli{at}lebs.cnrs-gif.fr.

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