Affinity Modification of Phenylalanyl-tRNA Synthetase from Thermus thermophilus by tRNA^Phe Transcripts Containing 4-Thiouridine

N. A. Moor^1*, V. G. Stepanov¹, 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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Photoreactive derivatives of tRNA^Phe containing residues of 4-thiouridine (s⁴U) were synthesized by the transcription system of T7 RNA polymerase. Complete substitution of s⁴U for 16 uridine residues ([16s⁴U]-tRNA^Phe) caused a 14-fold decrease in the catalytic efficiency of aminoacylation of the tRNA^Phe transcript by phenylalanyl-tRNA synthetase from T. thermophilus. [1s⁴U]-tRNA^Phe obtained by random incorporation of s⁴U residues with further isolation of s⁴U-monosubstituted RNA molecules on an affinity gel has the same kinetic parameters in aminoacylation as the tRNA^Phe transcript. The s⁴U-containing tRNA^Phe transcripts were shown to bind covalently to phenylalanyl-tRNA synthetase, and the specificity of modification was demonstrated. The modification stoichiometry determined in this work suggests that the enzyme is a functional dimer. The modification labels both alpha- and beta-subunits of the enzyme, which has an oligomeric structure of alpha₂beta₂, and forms "cross-linking" products of subunits upon modification with [16s⁴U]-tRNA^Phe. The prevalence of modification of the alpha-subunit suggests that tRNA has contacts with the enzyme, which have not been deciphered previously by X-ray analysis.

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

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