Modified constructs of the tRNA T Psi C domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases

The T Psi C stem and loop (TSL) of tRNA contains highly conserved nucleosid e modifications, m(5)C(49), T-54, Psi(55) and m(1)A(58). U-54 is methylated to m(5)U (T) by m(5)U(54) methyltransferase (RUMT); A(58) is methylated to m(1)A by m(1)A(58) tRNA methyltransferase (RAMT), RUMT recognizes and meth ylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RA MT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and syn thesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T-54 an d Psi(55)), naturally occurring modifications at unnatural positions (m(5)C (60)), altered sugar puckers (dU(54) and/or dU(55)) or with disrupted U-tur n interactions (m(1)Psi(55) or m(1)m(3)Psi(55)), The unmodified heptadecame r TSL was a substrate of both RAMT and RUMT. The presence of T-54 increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate, Local conformation around U-54 was found to be an important determinant for the activities of both RAMT and RUMT.