pre-tRNA End Processing

真核生物的tRNAs作为前体转录，在氨酰化和翻译之前必须在两端进行加工。5‘末端前导被RNaseP切割，3’末端拖尾可被3‘-tRNase内切去除。最后，tRNA核苷酸转移酶(NTase)将CCA添加到3‘-tRNase留下的3’端。这些tRNA末端加工酶的同源物已经在果蝇中被鉴定和部分鉴定。我们建议利用生物学和完全测序的果蝇基因组，研究所有三种酶的前tRNA末端加工，如下： 目的1.将使用RNAi这一强大的、创新的工具来抑制体内的基因表达 针对RNase P和3‘-tRNase，研究这些酶的核型和线粒体形式的反应级数和特性。 目的2.3‘-tRNase反决定簇假说(即tRNA在成熟的3’端带有CCA，不会通过3‘-tRNase循环)将被进一步研究。 目的3.分析3‘-tRNase和NTase与底物、产物和底物类似物的化学计量复合体，以更好地了解底物识别、结合和催化所需的位点和结构域。 目的4.用X射线衍射法测定3‘-tRNase的游离结构和与tRNA的络合结构。 本研究对黑腹果蝇的前tRNA末端加工进行了协调、全面的研究。 通过与世界级科学家建立的合作网络，这是可能的。

Eukaryotic tRNAs are transcribed as precursors and must be processed at both ends before aminoacylation and translation. A 5' end leader is cleaved off by RNase P and a 3' end trailer can be endonucleolytically removed by 3'-tRNase. Finally, tRNA nucleotidyltransferase (NTase) adds CCA to the 3' end left by 3'-tRNase. Homologs of these tRNA end-processing enzymes have been identified and partially characterized in Drosophila melanogaster. We propose to investigate pre-tRNA end-processing by all three enzymes, taking advantage of the biology and fully sequenced Drosophila genome, as follows: Aim 1. RNAi, a powerful, innovative tool for knocking down gene expression in vivo, will be used against RNase P and 3'-tRNase to investigate reaction order and identity of nuclear and mitochondrial forms of these enzymes. Aim 2. The 3'-tRNase anti-determinant hypothesis (that tRNA with CCA at its mature 3' end does not recycle through 3'-tRNase) will be further investigated. Aim 3. Stoichiometric complexes of 3'-tRNase and NTase with substrates, products and substrate analogs will be analyzed to better understand the sites and domains required for substrate recognition, binding and catalysis. Aim 4. The solution of 3'-tRNase structure, both free and complexed with tRNA, will be undertaken by X-ray diffraction crystallography. This coordinated, comprehensive study of D. melanogaster pre-tRNA end-processing is made possible by an established network of collaborations with world-class scientists.