The long term goal of this project is to define all the biochemical steps of tRNA processing and to examine their roles in the cell. Despite more than 40 years of work on tRNA, we still do not have a complete picture of this pathway. Current work is focused on tRNA modifications, which comprise the bulk of unknown biochemical steps. The number of tRNA molecules in the yeast cell dwarfs that of any other class of polynucleotide, accounting for 10 times more molecules than rRNA. There are a total of 22 different modifications in yeast tRNAs, catalyzed by multiple gene products. Many of these modifications are highly conserved, biochemically uncharacterized, unassigned to genes, and of unknown role. Recent efforts in the field have led to the identification of a number of gene products involved in modification, using a combination of genetic, biochemical, and bioinformatic methods. Use of a biochemical genomics approach led to the identification of five gene products involved in four yeast modification activities. This has led to the beginnings of an understanding of the specificity, mechanism and function of many of these gene products, but leaves many unanswered questions. This work on tRNA processing will be continued by studying novel and interesting aspects of several tRNA modification enzymes, and by identifying new genes encoding modification activities.
Four specific aims are proposed. First, the biochemistry and biology of tRNA His guanylyltransferase will be studied. tRNA His species from all studied organisms are different from other tRNAs in having an extra G residue at the -1 position, which is added post-transcriptionally in eukaryotes. Two gene products that co-purify with tRNA His guanylyltransferase activity have been identified; one is essential and required for activity in vivo. The identities and activities of components of tRNA His guanylyltransferase will be determined, as well as the basis for tRNA recognition by the enzyme. Second, m1G9 methyltransferase will be examined to determine important catalytic and binding residues, because this enzyme is different from other known enzymes of this type. Third, several other modification enzymes and their corresponding genes will be identified. Some 17 modification enzymes have not yet been assigned to genes. Many of these modifications are biochemically interesting and are located in important regions of the tRNA. Finally, the function of some tRNA modification enzymes will be studied in vivo.
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