RNA splicing is an essential step in the expression of split genes encoding mRNA, rRNA and tRNA species. Splicing of these RNA precursors probably requires a minimum of three distinct splicing systems. Identification of the structural elements recognized by each of these splicing systems is essential to understanding the control of gene expression through selective RNA splicing. Among the classes of RNA splicing reactions in yeast, the tRNA splicing mechanism is one which has been studied at the biochemical level. The isolated splicing components and the reaction mechanism have been well characterized. This information provides a unique opportunity to examine in vitro the elements of pre-tRNA structure important in splicing. The experiments contained in this proposal can be summarized as follows: 1) In vitro mutagenesis of a yeast tRNATyr gene will be used to generate a series of variants in which small, defined segments of the gene are replaced by an oligonucleotide linker; 2) The original and variant tRNA genes will be joined to a phage SP6 promoter for in vitro transcription; and 3) The accuracy and efficiency of splicing will be compared for each of the pre-tRNA transcripts using purified yeast tRNA splicing enzymes and processing extracts. The results of these experiments will provide a systematic analysis of the effect of tRNA gene alterations on splicing of the resulting pre-tRNAs.