ENZYMATIC FORMATION OF MODIFIED NUCLEOSIDES IN TRANSFER-RNA - DEPENDENCE ON TRANSFER-RNA ARCHITECTURE

Information is still quite limited concerning the structural requireme nts in tRNA molecules for their post-transcriptional maturation by bas e and ribose modification enzymes. To address this question, we have c hosen as the model system yeast tRNA(Asp) that has a known three-dimen sional structure and the in vivo modifying machinery of the Xenopus la evis oocyte able to act on microinjected tRNA precursors. We have syst ematically compared the modification pattern of wild-type tRNA(Asp) wi th that of a series of structural mutants (21 altogether) altered at s ingle or multiple positions in the D-, T- and the anticodon branch, as well as in the variable region. The experimental system allowed us to analyze the effects of structural perturbations in tRNA on the enzyma tic formation of modified nucleosides at 12 locations scattered over t he tRNA cloverleaf. We found that the formation of m(1)G(37) and Psi(4 0) in the anticodon loop and stem and Psi(13) in the D-stem, were extr emely sensitive to 3D perturbations. In contrast, the formation of T-5 4, Psi(55) and m(1)A(58) in the T-loop, m(5)C(49) in the T-stem and m( 2)G(6) in the amino acid accepting stem were essentially insensitive t o change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA doma ins. The formation of m(2)G(26) at the junction between the anticodon and the D-stem, of Q(34) and manQ(34) in the anticodon loop were sensi tive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recogni tion by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to thei r sensitivities to structural perturbations in tRNA, is proposed. (C) 1996 Academic Press Limited