Poster abstracts

Poster number 41 submitted by Edward Miracco

Characterization of the products of 5-fluorouridine after the action of E. coli pseudouridine synthase TruB

Edward J. Miracco (University of Louisville), Shi Bai (University of Delaware), Eugene G. Mueller (University of Louisville)

Abstract:
Pseudouridine synthases (&psi S) are the enzymes responsible for the conversion of U to &psi, with most &psi Ss specific for particular uridine residues. The most highly conserved &psi is located in the T &psi C loop of tRNA, and it is generated by TruB and its orthologs. Unlike many &psi Ss that are potently inhibited by RNA containing 5-fluorouridine ([f⁵U]RNA), TruB handles [f⁵U]RNA as a substrate, turning f⁵U into two hydrated products in a time frame similar to the natural conversion of U to &psi . The two products of f⁵U have been isolated after digestion with nuclease S1 and alkaline phosphatase and characterized by mass spectrometry and a set of 1D and 2D NMR experiments. The products are both dinucleotides, and both are rearranged isomers of f⁵U that have been hydrated. In agreement with the cocrystal structure of TruB and [f⁵U]RNA, the more abundant product is almost certainly (5S,6R)-5-fluoro-6-hydroxy-pseudouridine.

References:
Hoang, C., and Ferre-D’Amare, A. R. (2001) Cocrystal Structure of a tRNA Psi55
Pseudouridine Synthase Nucleotide Flipping by an RNA-Modifying Enzyme.,
Cell 107, 929-939.

Spedaliere, C. J., and Mueller, E. G. (2004) Not all pseudouridine synthases are
potently inhibited by RNA containing 5-fluorouridine, RNA 10, 192-199.

Spedaliere, C. J., Ginter, J. M., Johnston, M. V., and Mueller, E. G. (2004) The
Pseudouridine Synthases: Revisiting a Mechanism That Seemed Settled, JACS 126, 12758-12759.

Keywords: pseudouridine, fluorouridine, NMR