THE DYNAMIC NMR STRUCTURE OF THE T-PSI-C-LOOP - IMPLICATIONS FOR THE SPECIFICITY OF TRANSFER-RNA METHYLATION

tRNA (m(5)U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethio nine-dependent methylation of uridine-54 in the T Psi C-loop of all tr ansfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA. structures, residue 54 is completely buried and the ques tion arises as to how RUMT gains access to the methylation site. A 17- mer RNA hairpin consisting of nucleotides 49-65 of the T Psi-loop is a substrate for RUMT Homonuclear NMR methods in conjunction with restra ined molecular dynamics (MD) methods were used to determine the soluti on structure of the I7-mer T-arm fragment. The loop of the hairpin exh ibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation wh ere a molecule exists in predominantly one major conformation. However , when resorting to softer refinement methods such as MD with time-ave raged restraints, the conflicting restraints in the loop can be satisf ied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely ex posed. The flexibility of the T Psi-loop in solution in conjunction wi th extensive binding studies of RUMT with the T Psi C-loop and tRNA su ggest that the specificity of the RUMT/tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-l oops, leading to a melting of the T-arm structure and making U54 avail able for methylation.