Splicing is a general phenomenon of eukaryotic RNA biosynthesis in all classes of RNA. The purpose of this investigation is to determine the extent to which pre-tRNA structure is important in the splicing reactions. First, our observation that pre-tRNA structure is dependent on Mg2+ and polyamine concentrations will be correlated with the effect these cations have on the specific activity of various yeast pre-tRNAs in splicing reactions. This correlation should provide us with a measure of the importance of intact pre-tRNA structure to recognition of the pre-tRNAs by the splicing enzymes. Second, the effect of altering the structure or sequence of a precursor on its function in the splicing reaations will be studied. The splicing reactions involve endonucleolytic excision of the intron and ligation of the resultant fragments (hereafter called halves) to yield the mature-sized tRNA. It is known that excision of the intron leaves a 2',3'-cyclic phosphate on the 5'-half and a 5'-hydroxyl on the 3'-half. The ligation reaction proceeds with phosphorylation of the 5' terminus of the 3'-half, opening of the 2',3'-cyclic phosphate to leave a 2'-phosphate on the 5'-half, and finally covalent joining of the 3'-hydroxyl terminus of the 5'-half to the 5'-phosphoryl terminus of the 3'-half. Additional structural or sequence requirements have yet to be determined. Pre-tRNAs or splicing intermediates that have been chemically modified will be tested to determine whether the modification affects the activity of the RNA in any of the splicing reactions. Synthetic model substrates will be made (a) by means of chemical and enzymatic generation of tRNA fragments and enzymatic RNA synthesis technology and (b) by means of directed in vitro mutagenesis of cloned yeast tRNA genes using chemically synthesized oligodeoxynucleotides. Among the proposed model substrates are molecules which will test (1) the role of the 2',3'-phosphate on the 5'-half in the ligation reaction; (2) the sequence and/or structure in the anticodon loop-intron region required by the splicing endonuclease, and (3) elements of the tRNA portion of the pre-tRNA that are necessary for splicing. These combined experiments should provide further insight into the mechanism of tRNA splicing and the structure of yeast tRNA precursors that is recognized by the splicing enzymes.
