Interaction between Yeast tRNAVal and Yeast Valyl-tRNA Synthetase Studied by Monochromatic-Ultraviolet-Light-Induced Cross-Linking

Abstract

The interaction between yeast tRNAVal and yeast valyl-tRNA synthetase was studied using a modified form of the photochemical cross-linking method introduced by Schoemaker and Schimmel [J. Mol. Biol. 84 (1974) 503–513]. Covalent complexes were obtained after irradiation by monochromatic light at 248 nm of mixtures of tRNA and enzyme at pH 7.2; they were separated from unbound tRNA and synthetase by high-voltage electrophoresis on sucrose gradients. After hydrolysis of the covalent complexes by ribonuclease T1, The free oligonucleotides, separated from the enzyme-oligonucleotide covalent complexes on Biogel P200, were fractionated on DEAE-cellulose columns. By comparison with the elution profile of a control tRNAVal, also submitted to irradiation but not cross-linked to the enzyme, the missing oligonucleotides joined to valyl-tRNA synthetase were identified, Six oligonucleotides were found, located on both sides of the amino-acid-accepting stem (U3-G7 and A69-A77oh, in the dihydrouracil stem and loop (U11-G15 and D20-G24), in the 5′ part of the anticodon arm (C26-G31) and in the anticodon loop (A36-G40). After irradiation of complexes, where the tRNA was specifically labelled at the 3′-terminal adenosine, it was shown by a direct method that, in agreement with the indirect cross-linking method, the 3′-terminal ribo-nuclease T1 oligonucleotide (A69-A77oh) was joined to the enzyme, and that this joining occurred by at least two linkages, one involving the 3′-terminal adenosine, This last linkage, however, occurs at a rather poor efficiency (20%) as compared to the joining of the complete oligonucleotide (60%). When the cross-linked regions are pictured in the three-dimensional model of tRNA it appears that they are all located at the inside of the two branches forming the L-shaped tRNA structure. This geometry of interaction of tRNAVal with valyl-tRNA synthetase is similar to that already reported for other systems [Rich and Schimmel, Nucletc Acid Res. 4 (1977) 1649-1665] and may thus represent a general mode of interaction of tRNAs with synthetases.