Effect of intramolecular photochemical cross-linking and of alkylation of 4-thiouridine in E. colitRNA_val1: On the heterologous mischarging by yeast phenylalanyl-trna synthetase

The ability of yeast phenylalanyl-tRNA synthetase to carry out the heterologous mischarging of nine E. coli tRNAs with phenylalanine, and the presence of a common sequence in these tRNAs in the double stranded region adjacent to the dihydrouridine loop, has led to the proposal (by Dudock) that this region of the tRNA is involved in recognition by the yeast enzyme. The validity of this hypothesis has now been examined by chemical modification of the region in question using as a test tRNA, E. coli tRNA_val1Photochemical cross-linking of₄S₍₈₎ and C₍₃₎ by irradiation at 335 nm led to a complete loss of the ability of yeast phenylalanyl-tRNA synthetase to functionally recognize tRNA_1Val and the rate of cross-linking was correlated with the rate of loss of activity when appropriate assay conditions were used. Cross-linking had no effect on the recognition by the homologous E. coli valyl-tRNA synthetase [EC 6.1.1. 9].Similarly, S-alkylation of the ₄S₍₈₎ residue with iodoacetamide at pH 9 yielded the uridine-4-thio(2-acetamide) derivative of tRNA with no loss of homologous recognition but with complete loss of heterologous charging activity.These results provide evidence that at least part of the yeast phenylalanyl-tRNA synthetase recognition site is located in the region of the tRNA proposed by Dudock, and, as a corollary, show that the E. coli valyl-tRNA synthetase recognition site(s) must be elsewhere in the molecule.