All living organisms contain transfer ribonucleic acids (tRNA) which function as an integral part of the protein synthesizing machinery and play a role in regulation of gene expression. Of all the RNA populations, tRNAs have the most extensive and varied post-transcriptional modifications. During normal and abnormal cellular development, differences occur in the modified nucleotide content of specific tRNAs. Although the biosynthesis of many modified nucleotides entails post-transcriptional modification of precursor tRNAs, the function of most modified nucleotides is unknown.
The specific aim of this proposal is to determine the mechanism by which selected modified nucleotides modulate tRNA function. Our approach is to obtain homogenic tRNAs and compare their kinetics in the various tRNA requiring reactions. Initially, we will use the naturally occurring homogenic mammalian tRNAs we have isolated from various normal and tumor tissues. Concurrently, we will clone the mammalian phenylalanine tRNA genes and develop an in vitro system by which we can transcribe cloned tRNA genes and use these primary transcripts to obtain a series of in vitro processed homogenic tRNAs. We will compare the reaction kinetics of these naturally occurring and in vitro produced homogenic tRNAs using our complete and fractionated mammalian elongator tRNA dependent in vitro protein synthesis systems. We are in a unique position to accomplish these goals because we have extensive experience in isolating and sequencing mammalina tRNAs, in purifying and characterizing enzymes involved in post-transcriptional tRNA modification, in cloning and sequencing eucaryote mitochondrial tRNA genes, and in developing mammalian cytoplasmic elongator tRNA dependent in vitro protein synthesis systems. These studies will deepen our understanding of the mechanism of the mammalian nuclear-cytoplasmic tRNA processing systems and will allow us to determine directly the effect of alterations in a tRNA's modified nucleotide content during normal and abnormal cellular development.
