Effect of Nucleotide Replacements in tRNA^Phe on Positioning of the Acceptor End in the Complex with Phenylalanyl-tRNA Synthetase

I. A. Vasil'eva¹, A. Favre², O. I. Lavrik¹, and N. A. Moor^1*

¹Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk 630090, Russia; fax: (8-383) 233-3677; E-mail: moor@niboch.nsc.ru

²Institute Jacques Monod, CNRS-Universite Paris 7-Universite Paris 6, 75251 Paris Cedex 05, France

^* To whom correspondence should be addressed.

Received April 25, 2003; Revision received July 2, 2003

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The effect of replacement of tRNA^Phe recognition elements on positioning of the 3´-terminal nucleotide in the complex with phenylalanyl-tRNA synthetase (PheRS) from T. thermophilus in the absence or presence of phenylalanine and/or ATP has been studied by photoaffinity labeling with s⁴U76-substituted analogs of wild type and mutant tRNA^Phe. The double mutation G34C/A35U shows the strongest disorientation in the absence of low-molecular-weight substrates and sharply decreases the protein labeling, which suggests an initiating role of the anticodon in generation of contacts responsible for the acceptor end positioning. Efficiency of photo-crosslinking with the alpha- and beta-subunits in the presence of individual substrates is more sensitive to nucleotide replacements in the anticodon (G34 by A or A36 by C) than to changes in the general structure of tRNA^Phe (as a result of replacement of the tertiary pair G19-C56 by U19-G56 or of U20 by A). The degree of disorders in the 3´-terminal nucleotide positioning in the presence of both substrates correlates with decrease in the turnover number of aminoacylation due to corresponding mutations. The findings suggest that specific interactions of the enzyme with the anticodon mainly promote the establishment (controlled by phenylalanine) of contacts responsible for binding of the CCA-end and terminal nucleotide in the productive complex, and the general conformation of tRNA^Phe determines, first of all, the acceptor stem positioning (controlled by ATP). The main recognition elements of tRNA^Phe, which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm.

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KEY WORDS: acceptor end of tRNA, affinity modification, 4-thiouridine, phenylalanyl-tRNA synthetase, Thermus thermophilus, tRNA recognition

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