Enzymatic Formation of Modified Nucleosides in tRNA: Dependence on tRNA Architecture

Information is still quite limited concerning the structural requirements in tRNA molecules for their post-transcriptional maturation by base and ribose modification enzymes. To address this question, we have chosen as the model system yeast tRNA Aspthat has a known three-dimensional structure and the in vivomodifying machinery of the Xenopus laevisoocyte able to act on microinjected tRNA precursors. We have systematically compared the modification pattern of wild-type tRNA Aspwith that of a series of structural mutants (21 altogether) altered at single 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 enzymatic formation of modified nucleosides at 12 locations scattered over the tRNA cloverleaf. We found that the formation of m 1G 37and Ψ 40in the anticodon loop and stem and Ψ 13in the D-stem, were extremely sensitive to 3D perturbations. In contrast, the formation of T 54, Ψ 55and m 1A 58in the T-loop, m 5C 49in the T-stem and m 2G 6in the amino acid accepting stem were essentially insensitive to change in the overall tRNA architecture; these modified nucleosides were also formed in appropriate minimalist (stems and loops) tRNA domains. The formation of m 2G 26at the junction between the anticodon and the D-stem, of Q 34and manQ 34in the anticodon loop were sensitive only to drastic structural perturbation of the tRNA. Altogether, these results reflect the existence of different modes of tRNA recognition by the many different modifying enzymes. A classification of this family of maturation enzymes into two major groups, according to their sensitivities to structural perturbations in tRNA, is proposed.