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.

在单细胞泡菜的大核核糖体RNA基因中， 四膜热嗜热，26S rRNA编码区被413中断 碱基对中间序列（IVS）。 IVS包含在 主转录本，但随后通过剪接从RNA中切除。 我们最近开发了一个完全定义的体外剪接系统。 我们发现，剪接活性与IVS的IV部分固有 RNA，不需要酶或其他蛋白质。 目的 拟议的研究是为了进一步表征前rNNA的机制 剪接和RNA的结构需要介导反应。 这 外显子连接期间形成的结构（rRNA 与IVS接壤的序列）将由RNA指纹识别确定，并且 该连接反应的动力学和辅助因子要求将是 调查。 RNA中鸟氨酸辅因子的结合位点将 具有鸟苷类似物的动力学研究的特征和 光性标签。 IV的二级结构将是 使用化学和酶探针，计算机计算以及 序列的系统发育比较。 切除的IVS RNA的结构 当它是前体的一部分时，将与IVS的相比。 重组质粒DNA的广义和位置诱变 其次是体外转录将用于产生改变的前rNNA 将在体外剪接系统中测试的分子；这会 允许在剪接反应的特定步骤中存在缺陷 与RNA序列的特定变化。 最后，一般性 将研究四膜前rNNA剪接机制。 这 四膜虫前RRNA剪接系统异常适合详细 学习。 希望某些发现将适用于 人类mRNA剪接的易于处理的问题要少得多，其中缺陷 已知会引起诸如β+thalassysya之类的疾病。