TRANSFER RNA(PHE) ISOACCEPTORS POSSESS NONIDENTICAL SET OF IDENTITY ELEMENTS AT HIGH AND LOW MG2+ CONCENTRATION

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of II positions known as identity elements f or E. coli phenylalanyl-tRNA synthetase (FRS), In order to reveal stru ctural requirements for FRS recognition, aminoacylation of wild-type p hage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript, The wild-type phage T5 transcr ipt can be aminoacylated with the same catalytic efficiency as the E. coli counterpart, However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 an d 15 mM, respectively, Aminoacylation assays with tRNA(Phe) mutants re vealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation a t 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in p hage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcri pt exhibits virtually no influence on aminoacylation rate at 15 mM Mg2 +; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrati ons; (iv) phage transcripts with double substitutions A20U+A35C and A2 0U+A36C were very poor substrates for FRS, Collectively, the results i ndicate the non-identical made of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity el ements, the local tRNA conformation is essential for recognition by FR S. (C) 1997 Federation of European Biochemical Societies.