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Nucleic Acids Research, 2000, Vol. 28, No. 6 1374-1380
© 2000 Oxford University Press

Modified constructs of the tRNA T{Psi}C domain to probe substrate conformational requirements of m1A58 and m5U54 tRNA methyltransferases

Ranjita Sengupta, Saulius Vainauskas1, Connie Yarian, Elzbieta Sochacka2, Andrzej Malkiewicz2, Richard H. Guenther, Karl M. Koshlap and Paul F. Agris*

Department of Biochemistry, North Carolina State University, 128 Polk Hall, Box 7622, Raleigh, NC 27695-7622, USA, 1Department of Biochemistry and Biophysics, Vilnius University, Vilnius 2009, Lithuania and 2Institute of Organic Chemistry, Technical University, Lodz 90-924, Poland

The T{Psi}C stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m5C49, T54, {Psi}55 and m1A58. U54 is methylated to m5U (T) by m5U54 methyltransferase (RUMT); A58 is methylated to m1A by m1A58 tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T54 and {Psi}55), naturally occurring modifications at unnatural positions (m5C60), altered sugar puckers (dU54 and/or dU55) or with disrupted U-turn interactions (m1{Psi}55 or m1m3{Psi}55). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T54 increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U54 was found to be an important determinant for the activities of both RAMT and RUMT.

* To whom correspondence should be addressed. Tel: +1 919 515 6188; Fax: +1 919 515 2047; Email: agris@bchserver.bch.ncsu.edu Present address: Ranjita Sengupta, Department of Molecular and Cell Biology, 401 Barker Hall, University of California at Berkeley, Berkeley, CA 94720, USA


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