INTERACTION BETWEEN YEAST TRANSFER RNAVAL AND YEAST VALYL-TRANSFER RNA-SYNTHETASE STUDIED BY MONOCHROMATIC-ULTRAVIOLET-LIGHT-INDUCED CROSS-LINKING

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. Copyright © 1979, Wiley Blackwell. All rights reserved