The long term objective is to understand the mechanism of catalysis and substrate recognition of tRNA (m5U54)-methyltransferase (RUMT). A secondary objective is to initiate work on tRNA pseudo uridine synthase I and II (II, psi55 and I, hisT). The specific aims are summarized as follows: (1) We will study the conformational changes that occur in tRNA and the T arm of tRNA on binding to RUMT. Rapid kinetics will be probed using stopped flow fluorescence quenching. Mutagenesis of the RNA substrate will be aimed at destabilizing secondary and tertiary RNA structure, and the effect on catalysis by RUMT will be destabilizing secondary and tertiary RNA structure, and the effect on catalysis by RUMT will be assessed. In appropriate collaborations, NMR and X-ray crystallography will be performed on the enzyme and substrate, individually and in complex. (2) We will study aspects of tRNA recognition by RUMT. RNA footprinting techniques will be used to identify RUMT-RNA contacts outside of the T arm. Chemical synthesis of RNA analogs will be used to obtain substrates with various functional group substitutions, such as deoxyribose at specific positions. In vitro selection (SELEX) will be used to identify "best binding' sequences. (3) We will attempt to crystallize RUMT and RUMT-RNA complexes for future X-Ray structure determination. (4). We will determine whether RNAs other than tRNA are substrates for RUMT. (5) Studies will be performed with Pseudo Uridine (psi55) Synthase II and Pseudo Uridine Synthase I (his T), closely following the specific aims of our proposed studies of RUMT. This work is significant at several different levels of biomedical research. First, the research seeks to understand more about enzyme catalysis, providing insight into how such reactions occur in the complex environment of an RNA molecule. Second, the work attempts to identify elements contributing to protein-RNA recognition and to uncover general rules by which certain proteins recognize common structural features of RNA. The work also seeks to identify conformational changes of tRNA which accompany protein recognition, and to initiate structural studies on unique RNA-protein complexes. Third, if other RNAs are potential substrates for RUMT (or psi 55 synthase), the mutagenesis studies performed here will assist in their identification. Finally, some effects of the anti-cancer agent FUra may be due to its incorporation into RNA. As work in this area progresses, this point will become clarified and could lead to the identification and exploitation of new drug targets.