Localization of the Binding Site for the 3´-Terminal Sequence of tRNA^Phe in Subunits of Phenylalanyl-tRNA Synthetasefrom Thermus thermophilus

N. A. Moor^1*, V. N. Ankilova¹, A. Favre², and O. I. Lavrik¹

¹Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090 Russia; fax: 8-3832343659; E-mail: moor@niboch.nsc.ru

²Institut Jacques Monod, CNRS-Universite Paris 7, 75251 Paris Cedex 05, France

^* To whom correspondence should be addressed.

Received February 10, 1998; Revision received April 23, 1998

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A photoreactive tRNA^Phe derivative containing a 4-thiouridine residue at the 3´-end (tRNA^Phe-s⁴U-75) was prepared by tRNA nucleotidyltransferase-mediated incorporation of s⁴UMP into a tRNA^Phe transcript lacking the 3´-terminal dinucleotide. The resulting tRNA^Phe-s⁴U-75 was covalently bound to phenylalanyl-tRNA synthetase from Thermus thermophilus, and all criteria of an affinity modification were met. The main products of modification displaying various electrophoretic mobilities were formed by binding tRNA^Phe-s⁴U-75 to the beta-subunit (major) of the enzyme. These data suggest that the nucleotide found at position 75 of tRNA^Phe interacts with the beta-subunit of phenylalanyl-tRNA synthetase.

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KEY WORDS: affinity modification, 4-thiouridine, tRNA^Phe, phenylalanyl-tRNA synthetase, Thermus thermophilus

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