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

N. A. Moor1*, 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
Photoreactive derivatives of tRNAPhe 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]-tRNAPhe) caused a 14-fold decrease in the catalytic efficiency of aminoacylation of the tRNAPhe transcript by phenylalanyl-tRNA synthetase from T. thermophilus. [1s4U]-tRNAPhe 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 tRNAPhe transcript. The s4U-containing tRNAPhe 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]-tRNAPhe. 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.
KEY WORDS: affinity modification, 4-thiouridine, tRNAPhe, phenylalanyl-tRNA synthetase, Thermus thermophilus