SPLICING OF A RIBOSOMAL RNA PRECURSOR

核糖体 RNA 前体的剪接

• 批准号: 3275304
• 负责人: THOMAS ROBERT CECH
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275304
• 关键词: Escherichia coli; Tetrahymena; autoradiography; chemical fingerprinting; chemical group; cofactor; enzyme structure; evolution; gel electrophoresis; gene expression; genetic manipulation; genetic recombination; genetic strain; guanosine diphosphate; microorganism genetics; molecular cloning; nucleic acid sequence; nucleoside analog; radiotracer; ribosomal RNA; temperature sensitive mutant; tissue /cell culture

In the macronuclear ribosomal RNA genes fo the unicellular eucaryote, Tetrahymena thermophila, the 26S rRNA coding region is interrupted by a 413 base pair intervening sequence (IVS). The IVS is contained within the primary transcript but is subsequently excised from the RNA by splicing. We have recently developed a completely defined, in vitro splicing system. We found that the splicing activity is intrinsic to the IVS portion of the RNA, and that no enzyme or other protein is required. The objective of the proposed research is to further characterize the mechanism of pre-rRNA splicing and the structure of the RNA needed to mediate the reaction. The structure of the junction formed during ligation of the exons (rRNA sequences bordering the IVS) will be determined by RNA fingerprinting, and the kinetics and cofactor requirements of this ligation reaction will be investigated. The binding site of the guanosine cofactor in the RNA will be characterized by kinetic studies with guanosine analogs and by photoaffinity labeling. The secondary structure of the IVS will be determined using chemical and enzymatic probes, computer calculations and phylogenetic comparison of sequences. The structure of the excised IVS RNA will be compared to that of the IVS when it is part of the precursor. Generalized and sitespecific mutagenesis of recombinant plasmid DNA followed by in vitro transcription will be used to produce altered pre-rRNA molecules which will be tested in the in vitro splicing system; this will allow defects in specific steps of the splicing reaction to be correlated with specific changes in the RNA sequence. Finally, the generality of the Tetrahymena pre-rRNA splicing mechanism will be investigated. The Tetrahymena pre-rRNA splicing system is unusually amenable to detailed study. It is hoped that some of the findings will be applicable to the much less tractable problem of human mRNA splicing, defects in which are known to cause diseases such as Beta+-thalassemia.

在单细胞真核生物的大核核糖体RNA基因中， 嗜热四膜虫，26 S rRNA编码区被413个碱基中断， 碱基对间插序列（IVS）。 IVS包含在 初级转录物，但随后通过剪接从RNA切除。 我们最近开发了一个完全定义的体外剪接系统。 我们发现，剪接活性是固有的IVS部分， RNA，并且不需要酶或其他蛋白质。 的目的 拟议的研究是进一步表征前rRNA的机制， 剪接和介导反应所需的RNA结构。 的 外显子（rRNA）连接过程中形成的连接结构 与IVS接壤的序列）将通过RNA指纹图谱确定，并且 该连接反应的动力学和辅因子要求将是 研究了 RNA中鸟苷辅因子的结合位点将 通过鸟苷类似物的动力学研究表征， 光亲和标记 IVS的二级结构将是 使用化学和酶探针，计算机计算和 序列的系统发育比较。 切除的IVS RNA的结构 将与IVS作为前体的一部分进行比较。 重组质粒DNA的广义突变和定点突变 随后进行体外转录，将用于产生改变的前rRNA 将在体外剪接系统中测试的分子;这将 使剪接反应的特定步骤中的缺陷相互关联 在RNA序列上有特定的变化。 最后， 四膜虫前rRNA剪接机制将进行研究。 的 四膜虫前rRNA剪接系统是不寻常的服从详细 study. 希望其中一些调查结果将适用于 更不容易处理的问题，人类mRNA剪接，其中的缺陷是 已知会导致β +地中海贫血等疾病。