Isotope labeling of free and aminoacyl transfer RNA synthetase-bound transfer RNA.
The structural organization of the complex of Escherichia coli Ile-tRNA synthetase and tRNA Ile has been studied by isotope labeling of purine units in the tRNA. Free or bound tRNA is incubated in tritiated water for 5 to 15 h at 37 degrees in order to incorporate tritium into the C-8 positions of purine units. (Previous work has shown that the labeling rate of a purine is very sensitive to its microenvironment.) Under conditions where exchange-out does not occur, the nucleic acid is digested with nucleases and purines are subsequently isolated from known locations in the structure. Four purines are substantially perturbed by bound Ile-tRNA synthetase; in each case, the rate of labeling is retarded in the presence of the synthetase. The four purines occur at or near the 3' terminus and at the interface of the dihydrouridine stem and loop. These bases occur in segments of the tRNA that previous photochemical cross-linking studies have identified as important for synthetase-tRNA interactions. It appears that the effects observed on these sites are caused by their direct interaction with or shielding by the bound synthetase. In addition, two other sites, one in the anticodon and one in the amino acid acceptor-T psi C helix, appear to be perturbed (retarded labeling rates) by the bound enzyme. The data also suggest there is no significant conformational change in the tRNA upon binding to the synthetase.
D000227:Adenine Nucleotides / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D007532:Isoleucine; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007553:Isotope Labeling; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism*; D014316:Tritium
D000227:Adenine Nucleotides; D007532:Isoleucine; D014316:Tritium; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase