Interaction of T. thermophilus Phenylalanyl-tRNA Synthetase with the 3´-Terminal Nucleotide of tRNA^Phe

I. A. Vasil'eva, V. N. Ankilova, O. I. Lavrik, and N. A. Moor^*

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk, 630090 Russia; fax: 8-383-233-3677; E-mail: moor@niboch.nsc.ru

^* To whom correspondence should be addressed.

Received November 12, 1999; Revision received February 10, 2000

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The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3´-terminal nucleotide of tRNA^Phe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3´-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNA^Phe derivatives containing a photoreactive 4-thiouridine (tRNA^Phe-s⁴U-76) or 6-thioguanosine residue (tRNA^Phe-s⁶G-76) in the 3´-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3´-terminal adenosine in productive interaction of tRNA^Phe with the enzyme: tRNA^Phe-s⁴U-76 cannot be aminoacylated; the replacement of A-76 with s⁶G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased V_max value. Relative cross-linking of the s⁶G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNA^Phe-s⁴U-76. The dialdehyde derivative, tRNA^Phe-A_ox-76, with periodate-oxidized 3´-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNA^Phe-s⁶G-76. The results suggest specific binding of the 3´-terminal nucleotide of tRNA^Phe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNA^Phe acceptor end with PheRS. The correct positioning of the 3´-terminal nucleotide of tRNA^Phe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.

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KEY WORDS: acceptor end, affinity labeling, phenylalanyl-tRNA synthetase, tRNA, Thermus thermophilus

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