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.

真核生物 tRNA 被转录为前体。 5' 末端前导序列和 3' 末端尾随序列分别通过 RNase P 和 3'- tRNase 进行核酸内切去除。每个 tRNA 3' 端发现的 CCA 不是转录编码的，必须通过 tRNA 核苷酸转移酶分别添加到 3'-tRNase 产生的新 3' 端。每个 tRNA 3' 端发现的 CCA 不是转录编码的，必须通过 tRNA 核苷酸转移酶添加到 3'-tRNase 产生的新 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 环三级接触区域的相互作用不同（Levinger 等人，JBC 273：1015，1998）。 3' 末端 CCA 是真核 3'-tRNase 的抗决定簇（Mohan 等人，RNA 5:245, 1999）。中部受体茎中的序列和结构对于 3'-tRNase 来说似乎比 RNase P 更重要，并且 3'-tRNase 可能会接触 4/69 碱基对（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来饱和果蝇 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 的突变与多种遗传性疾病有关。人类线粒体 tRNA 中的突变 A7445G 与多种遗传性疾病有关。人线粒体 tRNA/Ser(UCN) 中的突变 A7445G 导致非综合征性耳聋（Guan 等人，MCB 18：5868，1998）。 A7445G 将紧随 3'-tRNase 切割位点 (->) 的 G->UCU 更改为 G->CCU，理论上会导致 3' 末端加工缺陷。我们假设这种取代过程产生了 3'-tRNase 抗决定簇。我们建议使用从培养的人类细胞中提取的线粒体 tRNA 加工提取物和 tRNA 前体的体外转录来表征假定的加工缺陷。