Book of Abstracts: Albany 2003
June 17-21 2003
Interaction of DMAPP:tRNA Prenyltransferase with Escherichia coli tRNAPhe
The modification of bases in tRNA and rRNA molecules occurs in all cells, yet relatively little is known about the fundamental aspects of this process, including the molecular basis for enzyme-RNA recognition, the mechanisms of catalysis used for modification, and the functions of the modified bases in RNA molecules. The mechanisms used by modification enzymes to discriminate between similarly shaped substrate and nonsubstrate tRNA molecules is of fundamental biochemical interest and importance. Although this problem has been investigated at the atomic level for the aminoacyl-tRNA synthetases, similar efforts have been made for only a handful of the tRNA base-modification enzymes.
We are using the enzyme DMAPP:tRNA prenyltransferase (MiaA) as a model system for examining the details of protein-RNA recognition, the mechanism of action of tRNA modification enzymes, and the functional relationships between tRNA modification and cellular metabolism. MiaA catalyzes the addition of a dimethylallyl unit from dimethylallyl diphosphate (DMAPP) to the exocyclic amino nitrogen (N6 of A37, the nucleotide flanking the 3' end of the anticodon in E. coli tRNAPhe to form i6A37. We have used enzymatic and chemical footprinting to probe the interaction of MiaA with tRNAPhe and are also using NMR spectroscopy to examine in detail the interaction of MiaA with the anticodon stem-loop. The unmodified anticodon stem-loop forms a compact tri-loop that is distinctly different from the crystal structure of yeast tRNAPhe. The enzyme binds tRNA as a dimer and contacts the anticodon and acceptor stems of tRNA. The TψC stem becomes accessible to nuclease V2 when complexed to MiaA. The NMR data indicate that the anticodon stem-loop is melted upon MiaA binding. We are also examining the ability of endogenous non-tRNA molecules to serve a substrates for MiaA. Since MiaA does not require the full-length tRNA for activity, other RNAs in the cell that contain the necessary recognition motif could serve as substrates but may be excluded from prenylation.