Structure Function Analysis of human m1A58 tRNA Methyltransferase
Final Report Abstract
Human m1A58 methyltransferase catalyzes the methylation of the N1 position of nucleoside A58 in the T-loop of many tRNAs. The methylation of A58 in tRNA3Lys, which also serves as a primer during HIV reverse transcription, has been reported to be essential for normal reverse transcription. In this study, I sought to determine the crystal structure of m1A58MT, which is a potential anti HIV-drug target, in complex with tRNA3Lys and cofactor to understand how the enzyme makes the T-loop accessible and how it becomes totally specific for tRNA A58. m1A58MT was recombinantly expressed in E.coli and purified to homogeneity. Formation of a heterotetramer, which is comprised of two subunits Trm6 and Trm61, was supported by biochemical studies. Full-length tRNA3Lys was produced by in vitro transcription. The purified enzyme was active against tRNA3Lys as shown in a fluorescent-based enzyme assay. Extensive crystal screening was carried out using combinations of different protein constructs, RNA and cofactor however no reproducible crystals could be obtained. In collaboration with the proteome-antibody center at UCSF Fab fragments against m1A58MT were generated for use in co-crystallization studies.
