Removal of intervening sequences (splicing) is a requirement common to the expression of all classes of genes. Among the four identified classes of splicing reactions, this research focuses specifically on splicing of tRNA precursors. Although similarities among the other classes of reactions suggest they may have a common origin, the unique features of the tRNA splicing reactions suggest a separate origin or early divergence for this class. Thus an analysis of tRNA splicing will shed light not only on the role of splicing in the expression of this essential class of genes but also on the potential origins of tRNA gene introns, their significance in the evolution of tRNA gene families and the relationship of tRNA splicing to other types of splicing reactions. Splicing of tRNA precursors in extracts of Saccharomyces cerevisae requires 3 distinct activities: a membrane-bound endonuclease, an NAD- dependent RNA ligase and an NAD-dependent phosphotransferase. The proposed experiments fall into two general categories. These are structure/function relationships in tRNA ligase, including the assembly of a splicing complex, and development of an inducible tRNA expression system. In the first project area, directed mutagenesis will be used to identify functional sites on the multifunctional .ligase polypeptide. Interactions among functional elements and substrate recognition will he examined both in vitro and in vivo. Localization of ligase within the nucleus and assembly of a splicing complex will be examined by immunofluorescence and immunogold electron microscopy. The second project area is the development of an inducible tRNA gene expression system to be used in analyzing splicing defects in vivo, ordering of events in a tRNA processing pathway and regulation of tRNA biosynthesis.
