EUKARYOTIC TRNA PROCESSING BY RNASE P AND 3' RNASE

通过 RNA酶 P 和 3 RNA酶进行真核 TRNA 加工

• 批准号: 6452772
• 负责人: LEVINGER LOUIS
• 金额: $ 3.15万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6452772
• 关键词: DNA footprinting; Drosophilidae; RNA biosynthesis; affinity chromatography; chemical synthesis; enzyme activity; eukaryote; gel mobility shift assay; gene mutation; genetic transcription; ion exchange chromatography; nucleic acid reconstitution; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing; ribonuclease III; ribonuclease P; site directed mutagenesis; small nuclear RNA; tissue /cell culture; transfer RNA

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.

真核TRNA被转录为前体。 RNase P和3'-trNase分别将5'End Leader和3'端拖车分别通过RNase p驱除。在每个tRNA的3'末端发现的CCA并未在转录编码上，必须通过tRNA核苷酸转移酶分别添加到由3' - trnase产生的新3'端。在每个tRNA的3'末端发现的CCA并未在转录编码中，必须通过tRNA核苷酸转移酶添加到由3'- trnase产生的新3'END中。我们已经研究了更改序列和结构对果蝇tRNA/他的最终处理的效率和动力学的影响。 RNase P和3'-trnase与普遍保守的D/T环第三纪触点的区域不同（Levinger等人，JBC 273：1015，1998）。 3'端CCA是真核3'-trnase的抗确定性（Mohan等，RNA 5：245，1999）。中等受体茎中的序列和结构对于3'-trnase似乎比RNase P更重要，而3'- TRNase可以接触4/69碱基对（Mohan and Levinger，手稿制备中）。我们建议通过两种类型的实验更完整地​​描述pre-tRNA终端处理的序列和结构要求。首先，我们希望用果蝇tRNA/他的D和T环饱和。我们将测试这些序列变化对终端处理效率和动力学以及tRNA结构的影响。其次，人线粒体TRNA中的突变与各种遗传性疾病有关。人线粒体TRNA中的突变A7445G与各种遗传疾病有关。人线粒体tRNA/ser（UCN）中的突变A7445g引起非同伴耳聋（Guan等，MCB 18：5868，1998）。 A7445G将g-> ucu更改为g-> ccu，该ccu立即在3'-trnase裂解位点（ - >）后立即引起3'末端处理缺陷。我们假设这种替代程序是一种3'- TRNase抗确定性。使用线粒体TRNA加工提取物中培养的人类细胞和tRNA前体的体外转录，我们建议表征假定的加工缺陷。