tRNA splicing is essential for the survival of some, if not all, eukaryotes. The mechanism of tRNA splicing is highly conserved in eukaryotes, and best understood in the yeast Saccharomyces cerevisiae. Superficially, tRNA splicing appears simple; an endonuclease excises the intron; a ligase joins the two half-molecules; and a 2'-dephosphorylating activity removes the 2'-phosphate left at the splice junction. Yet the mechanism of splicing, particularly of the last step, is much more complicated than one would imagine. Removal of the splice junction 2'-phosphatase, rather than being catalyzed by a phosphatase, is catalyzed by an NAD-dependent, 2'-phosphate specific phosphotransferase. A protein like this has never been described, yet it is likely essential in some, if not all, eukaryotes in order to make functional TRNA. Moreover, the phosphate from the splice junction of TRNA ultimately ends up as a cyclic phosphate on NAD, or on a molecule similar to NAD. Such a molecule has also not been previously described. The objects of this proposal are to identify the proteins catalyzing this unusual but important dephosphorylation step in TRNA splicing and to determine the structure (and mechanism of formation) of the unusual phosphotransfer product. %%% The process of processing newly synthesized transfer RNA (TRNA) in yeast is essential for the TRNA to act in the synthesis of a protein. A significant part of the processing involves editing out particular sequences of bases in the strand. This research involves an enzyme which closes a gap in the TRNA strand (much like the process by which train cars are linked up after the cars in the center have been removed). The enzyme has some novel properties which merit investigation to distinguish it from other ligating enzymes . The information resulting from these studies will be new and important since the research is original.
