EUKARYOTIC TRNA PROCESSING BY RNASE P AND 3' RNASE

真核生物的tRNA被转录为前体。5 '端前导序列和3'端尾随序列分别通过RNase P和3 '-tRNase进行内切核酸酶切去除。在每个tRNA的3 '端发现的CCA不是转录编码的，并且必须分别通过tRNA核苷酸转移酶添加到由3'-tRNase产生的新的3'端。在每个tRNA的3 '端发现的CCA不是转录编码的，并且必须通过tRNA核苷酸转移酶添加到由3'-tRNase产生的新的3'端。我们研究了改变序列和结构对果蝇tRNA/His末端加工效率和动力学的影响。RNase P和3'-tRNase与普遍保守的D/T环三级接触区不同地相互作用（Levinger等人，JBC 273：1015，1998）。3 '端CCA是真核生物3'-tRNase的反决定簇（Mohan et al.，RNA 5：245，1999）。中间受体茎中的序列和结构似乎对3'-tRNase比RNase P更重要，并且3'-tRNase可以接触4/69碱基对（Mohan和Levinger，手稿在准备中）。我们建议通过两种类型的实验来更完整地描述前tRNA末端加工的序列和结构要求。首先，我们希望用单核苷酸取代饱和果蝇tRNA/His的D和T环。我们将测试这些序列变化对末端加工效率和动力学以及tRNA结构的影响。其次，人类线粒体tRNA的突变与各种遗传性疾病有关。人类线粒体tRNA中的A7445G突变与多种遗传性疾病有关。人线粒体tRNA/Ser（UCN）中的突变A7445G引起非综合征性耳聋（Guan等人，MCB 18：5868，1998）。A7445G将G-> UCU改变为G-> CCU，其紧接在3'-tRNase切割位点（->）之后，理论上引起3 '末端加工缺陷。我们假设这种替换程序是3'-tRNase抗决定簇。使用线粒体tRNA加工提取物从培养的人类细胞和体外translation的tRNA前体，我们建议的特点假设的处理缺陷。

Eukaryotic tRNAs are transcribed as precursors. The 5' end leader and 3' end trailer are endonucleolytically removed by RNase P and by 3'- tRNase, respectively. CCA found at the 3' end of every tRNA is not transcriptionally encoded, and must be added by tRNA nucleotidyl transferase to the new 3' end produced by 3'-tRNase, respectively. CCA found at the 3' end of every tRNA is not transcriptionally encoded, and must be added by tRNA nucleotidyl transferase into the new 3'end produced by 3'-tRNase. We have investigated the effects of changing sequence and structure on the efficiency and kinetics of Drosophila tRNA/His end processing. RNase P and 3'-tRNase interact differently with the region of universally conserved D/T loop tertiary contacts (Levinger et al., JBC 273:1015, 1998). The 3' end CCA is an anti-determinant for eukaryotic 3'-tRNase (Mohan et al., RNA 5:245, 1999). Sequence and structure in mid-acceptor stem appear to be more important for 3'-tRNase than for RNase P, and 3'- tRNase may contact the 4/69 base pair (Mohan and Levinger, manuscript in preparation). We propose to more completely describe the sequence and structure requirements for pre-tRNA end processing through two types of experiments. First, we wish to saturate the D and T loops of Drosophila tRNA/His with single nucleotide substitutions. We will test the effect of these sequence changes on end processing efficiency and kinetics and on tRNA structure. Second, mutations in human mitochondrial tRNAs have been linked with various inherited diseases. The mutation A7445G in human mitochondrial tRNAs have been linked with various inherited diseases. The mutation A7445G in human mitochondrial tRNA/Ser(UCN) causes non-syndromic deafness (Guan et al., MCB 18:5868, 1998). A7445G, which changes G->UCU to G->CCU which immediately following the 3'-tRNase cleavage site (->), is theorized to caused a 3' end processing defect. We hypothesize that this substitution procedures a 3'- tRNase anti-determinant. Using a mitochondrial tRNA processing extract from cultured human cells and in vitro transcriptions of tRNA precursors, we propose to characterize the postulated processing defect.