pre-tRNA End Processing

真核tRNA作为前体转录，并且必须在氨酰化和翻译之前在两端加工。5'端前导序列被RNase P切割掉，3'端尾部可以被3 '-tRNase内切核酸去除。最后，tRNA核苷酸转移酶（NTase）将CCA添加到3 '-tRNase留下的3'末端。这些tRNA末端加工酶的同源物已经在果蝇中被鉴定和部分表征。我们建议研究所有三种酶的前tRNA末端加工，利用生物学和完全测序的果蝇基因组，如下所示： 目标1。RNAi是一种强大的，创新的工具，用于敲低体内基因表达， 针对RNase P和3 '-tRNase，研究这些酶的核和线粒体形式的反应顺序和同一性。 目标2.将进一步研究3 '-tRNase反决定簇假说（在其成熟3'端具有CCA的tRNA不会通过3 '-tRNase再循环）。 目标3：将分析3 '-tRNase和NTase与底物、产物和底物类似物的化学计量复合物，以更好地了解底物识别、结合和催化所需的位点和结构域。 目标4。游离的和与tRNA复合的3 '-tRNase结构的溶液将通过X射线衍射晶体学进行。 本文对D.进行黑腹前tRNA末端加工， 通过与世界级科学家建立的合作网络。

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.