9723045 Wolfe Transfer RNA is synthesized as a precursor in the nucleus where it undergoes a series of maturation steps that include the cleavage of 5' and 3' sequences, base modifications, the removal of intervening sequences, and the addition of the oligonucleotide CCA to the 3' terminus. Transfer RNA is then exported to the cytosol where additional bases are modified and the 3'CCA terminus is repaired. In the mitochondria, precursor tRNA undergoes a similar maturation process. Many of the components of the maturation pathway in both the nucleus and mitchondria have not yet been identified. This research will identify new components of the tRNA maturation pathway, examine the organization of these proteins in a putative tRNA maturation complex, and to study protein interactions important to efficient maturation of tRNA. Crosslinking and immunoprecipitation experiments will utilize known maturation proteins as anchors to isolate and identify other components of the tRNA maturation pathway. Since Trm1p, Mod5p and Cca1p function in the maturation of nuclear and mitochondrial pre-tRNA they will be utilized as anchors to biochemically isolate chemical amounts of tRNA maturation complexes from both organelles. Crosslinking techniques will allow the isolation of proteins that along with the anchor protein interact with pre-tRNA. Components of these complexes will be identified by peptide sequence analysis. Peptide sequence information will then be used to search the yeast genomic data base for the corresponding gene. A two hybrid selection will be employed to determine the pattern of interactions between the newly identified proteins in the tRNA maturation complex. Genetic experiments will be conducted that exploit the central role of Ccalp in tRNA metabolism. ATP(CTP): tRNA nucleotidyhxansferase (Ccalp) is the only tRNA maturation enzyme characterized thus far that is essential for maturation of all cellular tRNAs. Ccalp localizes to the nucleus, mitochondria, and cytosol where it catalyzes the addition of the oligonudeotides CCA to the 3' end of nuclear and mitochondrial precursor tRNA. In the cytosol Cca1p repairs CCA end-shortened tRNAs. A genetic synthetic lethal screen will be carried out to identify essential Ccalp interactions that occur in a specific cellular compartment but may not be detected by biochemical means. Experimental data obtained from these biochemical and genetic approaches will yield new information concerning the identity of tRNA maturation proteins, the organization of tRNA maturation complexes and the protein interactions essential to tRNA metabolism in the cell. Transfer RNA is synthesized as a precursor in the nucleus where it undergoes a series of maturation steps. In the mitchondria, precursor tRNA undergoes a similar maturation process. Many of the components of the maturation pathway in both the nucleus and mitchondria have not yet been identified. Data obtained from these biochemical and genetic experiments will yield new information concerning the identify of tRNA maturation proteins, the organization of tRNA maturation complexes and the protein interactions essential to tRNA metabolism in the cell.
