Synthetase recognition determinants of E-coli valine transfer RNA

We have studied the interactions between Escherichia coli tRNA(Val) and val yl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease Si and r ibonuclease V1, and by analysis of the aminoacylation kinetics of mutant tR NA(Val) transcripts. Valyl-tRNA synthetase specifically protects the antico don loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNA(Val) against cleavage by double- and si ngle-strand-specific nucleases. Increased nuclease susceptibility at the en ds of the anticodon- and T-stems in the tRNA(Val) ValRS complex is indicati ve of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleo tides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator b ase, position 73, and the anticodon stem also are recognized by ValRS. Repl acing wild-type A73 with G73 reduces the aminoacylation efficiency more tha n 40-fold. However, the C73 and U73 mutants remain good substrates for ValR S, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNA(Val) contains no other synthetase reco gnition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res.,25, 4883-4890] . In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduce s the aminoacylation efficiency 50-fold. This is apparently due to the rigi dity of the anticodon stem caused by the presence of five consecutive C:G b ase pairs, since the A29:U41 mutant is readily aminoacylated. Identity swit ch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to trans form E. coli tRNA(Phe) into an effective valine acceptor. Replacing the ant icodon stem of tRNA(Phe) with that of tRNA(Val), however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.