In eukaryotes, a number of alterations of the primary Pol III transcript must occur before the functional tRNA reaches the protein synthesis machinery. These alterations include 5' and 3' end trimming, base modification, splicing and nuclear-cytoplasmic transport. Any of these have the potential for regulating the rate of appearance of all or a subset of tRNAs. The potential also exists for complex interplay among these events. The objective of the research proposed in this request is to investigate the biosynthesis of yeast (Saccharomyces) transfer RNAs (tRNAs) which arise through precursors which contain intervening sequences and require splicing as an obligatory step of maturation. The focus will be the identification of the elements necessary to promote the efficient and accurate recognition, binding and cleavage of the pre-tRNA by the tRNA splicing endonuclease (endonuclease, hereafter). The goal is to answer the following questions: (1) what elements of sequence and/or structure are required for the recognition and processing of pre- tRNAs by the endonuclease?; (2) are factors other than the endonuclease required for efficient recognition and binding?; (3) could relative rates of cleavage efficiency be a means of regulating cytoplasmic appearance of certain tRNA species? All of the specific aims will eventually intertwine in the evaluation of the contribution of all elements in the yeast tRNA splicing complex to the efficiency and accuracy of the endonuclease. The approaches toward this intertwining will involve establishing baseline kinetic parameters of the cleavage reaction using an in vitro transcribed pre-tRNAPhe and the intact endonuclease. From this point, we will characterize the parameters of and purify a catalytically active fragment that has been released from the nuclear membrane by limited proteolysis. The kinetic properties of all nine intron-containing pre-tRNAs will be investigated to begin addressing the third question. In addition, specific mutants will be constructed to complement this investigation as well as to continue systematically testing features of the pre-tRNA structure that contribute to binding and cleavage events. Lastly, preliminary observations that other factors may contribute to the efficiency and stability of the endonuclease will be pursued through the development of an assay for detection of the factor(s) as well as their purification and the characterization of their contribution to the kinetic pathway of pre-tRNA cleavage.
