Study of the interaction between yeast tRNAphe and yeast phenylalanyl-tRNA synthetase by monochromatic ultraviolet irradiation at various wavelengths. Advantages and limits of the method.
The interactions between yeast tRNAphe and phenylalanyl-tRNA synthetase were studied by analysis of the covalent adducts obtained upon monochromatic ultraviolet irradiation at different wavelengths (248, 282, 292, 302 and 313 nm). The high extent of inactivation of phenylalanyl-tRNA synthetase, together with the partial modification of tRNA, as well as the peculiar instability of most of the covalent bonds formed upon irradiation constitute severe limitations to the use of the technique and to the interpretation of the results. These disadvantages led us to select an irradiation wavelength of 248 nm and to use only mild isolation procedures allowing a good recovery of the covalent adducts formed. Seven major tryptic peptides of the enzyme were found to be cross-linked to tRNAPhe whereas six major T1-oligonucleotides were covalently linked to the protein, among these, the three cross-linked oligonucleotides previously described by Shoemaker and Schimmel (J. Biol. Chem. 250 (1975) 4440-4444) in the same system. The difference in the number of covalently linked oligonucleotides is discussed in the light of the instability of the covalent linkages. The localization of the six oligonucleotides at the inside of the two branches forming the L-shaped tRNA molecule is similar to that observed in the yeast valine system (Renaud et al., Eur. J. Biochem. 101 (1979) 475-483) and is consistent with the interaction model previously described (Rich and Schimmel, Nucl. Acids Res. 4 (1977) 1649-1665 and Ebel et al. in Transfer RNA: structure, properties and recognition, (1979) pp. 325-343 Cold Spring Harbor Laboratory, NY). The occurrence of covalent cross-linking upon irradiation in the tryptophan absorption band (302 nm) strongly suggests the participation of this residue in the stabilization of the tRNA enzyme complex.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000528:radiation effects; D009843:Oligoribonucleotides / Q000528:radiation effects; D010084:Oxidation-Reduction; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism* / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014466:Ultraviolet Rays
D009842:Oligopeptides; D009843:Oligoribonucleotides; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase