EUKARYOTIC TRNA PROCESSING BY RNASE P AND 3' RNASE

真核生物的trna作为前体进行转录。5‘端前导体和3’端后导体分别被RNase P和3'- tRNase内核溶解去除。在每个tRNA的3‘端发现的CCA没有转录编码，必须通过tRNA核苷酸转移酶分别添加到3’-tRNase产生的新的3'端。在每个tRNA的3‘端发现的CCA没有转录编码，必须通过tRNA核苷酸转移酶添加到3’-tRNase产生的新的3'端。我们研究了序列和结构变化对果蝇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环三级接触区域的相互作用不同（Levinger et al., JBC 273:1015, 1998）。3‘端CCA是真核生物3’-tRNase的反决定因子（Mohan et al., RNA 5:245, 1999）。与RNase P相比，3'-tRNase在中间受体茎中的序列和结构似乎更重要，并且3'-tRNase可能与4/69碱基对接触（Mohan和Levinger，手稿正在准备中）。我们建议通过两种类型的实验更完整地描述前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。人类线粒体tRNA/Ser（UCN）突变A7445G导致非综合征性耳聋（Guan et al., MCB 18:5868, 1998）。A7445G将G->UCU转变为紧跟3‘- trnase切割位点（->）的G->CCU，理论上是造成3’端加工缺陷的原因。我们假设这个替换过程是一个3'- tRNase反行列式。利用从培养的人类细胞中提取的线粒体tRNA加工提取物和tRNA前体的体外转录，我们提出表征假设的加工缺陷。

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.