The long term objectives are (a) to understand the mechanism of catalysis and substrate recognition of certain RNA modification enzymes 5-methyl uracil (m5U) and 5-methyl cystosine (m5C) methyltransferases, and RNA pseudouridine synthases and (b) to express and characterize catalytically active human tRNA N-1 methyl adenine (m1A58) methyltransferase and assess its potential as a target for retroviral infections.
The specific aims are summarized as follows. (1) With tRNA m5U54 methyltransferase, we will complete studies on the mechanism of RNA recognition by tRNA m5U54 methyltransferase, we will clone and characterize the two remaining putative E. coli rRNA m5U methyltransferases, complete bioinformatics studies, and perform crystallization trials of several homologs. (2) With RNA m5C methyltransferases, we will characterize the complex of enzyme covalently bound to RNA containing fluorocytosine, determine the mechanism, and perform crystallization trials of several homologs. (3) With pseudouridine synthases, we will unequivocally determine the mechanism of reaction with RNA containing fluorouracil, complete studies on the mechanism of RNA recognition by tRNA U55 pseudouridine synthase, assess the mechanism of a multi-site specific enzyme, and perform crystallization trials of several homologs. (4) With mI A58 methyltransferase, we will clone and express the active enzyme and perform crystallization trials. This work is significant at several levels. First, the research seeks to understand more about the mechanism of enzymes which modify pyrimidine and purine bases and to understand how such reactions occur when the base is in the complex environment of an RNA molecule. Second, the research attempts to identify elements contributing to protein-RNA recognition and to uncover general rules by which certain proteins recognize their target sites. Third, we use an informatics-based approach coupled with expression screening to rapidly assess RNA modifying enzymes most amenable to crystallization for structural studies. Fourth, the RNA-effects of the anti-cancer agent 5-Fluorouracil may be due to its incorporation into RNA, and subsequent covalent inhibition of RNA modification enzymes. As work progresses, this point will become clarified and could lead to the identification and exploitation of new drug targets. Finally, the work seeks to isolate and characterize a host enzyme that may be a novel target for HIV.
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