Eukaryotic tRNAs have the sequence CCA added post-transcriptionally to their 3' ends by ATP a G added post-transcriptionally to its 5' end. These nucleotide end-addition reactions follow the endonucleolytic removal of a 3' end trailer by 3'-tRNase and of a 5' end leader by Rnase P. CCA is required at the 3' end of TRNAS for aminoacylation; 3' end cleavage and CA addition are essential reactions in eukaryotes because the 3 'end CCA is not encoded in eukaryotic tRNA genes. The addition of CCA to the tRNA 3' is catalyzed and templated by tNtase, a protein enzyme. The enzyme presumably has three nucleotide binding pockets (for CCA) and cleft to properly position the tRNA acceptor stem. Identity of tRNA as the substrate which fits the postulated tNtase cleft has not been completely established.
The specific aims of the proposal are 1) to prepare and test for substrate recognition by introducing mutations or deletions in the tRNA acceptor stem, and 3) purify and clone the Drosophila tNTase gene. This will complement a systematic and continuing investigation of the tRNA processing pathway.
| Levinger, L; Jacobs, O; James, M (2001) In vitro 3'-end endonucleolytic processing defect in a human mitochondrial tRNA(Ser(UCN)) precursor with the U7445C substitution, which causes non-syndromic deafness. Nucleic Acids Res 29:4334-40 |
