GROUP II INTRON RNA SPLICING AND MOBILITY

第二组内含子 RNA 剪接和迁移率

• 批准号: 6179507
• 负责人: ALAN M. LAMBOWITZ
• 金额: $ 40.22万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179507
• 关键词: DNA footprinting; RNA; RNA directed DNA polymerase; RNA splicing; Streptococcus lactis; endonuclease; enzyme mechanism; enzyme structure; enzyme substrate complex; gene targeting; intermolecular interaction; introns; microinjections; protein protein interaction; technology /technique development

The proposed research involves continued biochemical-genetics studies of the protein-dependent splicing and mobility reactions of group II introns. Group II intron mobility is mediated by intron-encoded reverse transcriptases that also function as maturases to promote splicing of the intron. During the current grant period, we elucidated the general nature of the protein- dependent splicing and mobility reactions, and developed tools for studying the reactions further. First, we showed that group II intron mobility ( homing ) occurs via target-DNA primed reverse transcription mechanisms involving a novel intron-encoded DNA endonuclease, which is an RNP complex containing the intron- encoded protein and the excised intron RNA. In one mechanism, the intron RNA in the RNP complex reverse splices directly into the DNA target site and is then reverse transcribed by the intron-encoded protein. Second, we showed that group II intron endonucleases, which determine the site of intron insertion, use both their RNA and protein subunits to recognize specific sequences in their DNA target sites. In the case of the intron RNA, this involves base pairing between a 13-nt region of the DNA target site and sequences in the intron RNA and consequently, it is possible to change the specificity of the endonuclease simply by modifying the intron RNA. These findings raise the possibility that group II introns might be used as tools for targeted gene disruption and insertion of genetic information at desired sites in DNA genomes. Finally, we developed an efficient E. coli expression system for the Lactococcus lactis group II intron L1.LtrB. Recent extensions of the work include the development of genetic systems for studying both the retrohoming and splicing of the Lactococcal intron in E. coli, and methods for studying the splicing and mobility reactions using purified components. Specific aims are: (1) To continue biochemical analysis of the maturase-dependent group II intron splicing reaction. (2) To identify additional protein factors involved in group II intron splicing. (3) To analyze the reaction mechanism and DNA target-site interactions of the intron-encoded DNA endonuclease activity. (4) To support collaborations to determine X-ray crystal structures of the group II intron-encoded protein and its complexes with intron RNA and RT substrates. (5) To continue collaborations on group II intron mobility mechanisms in yeast mitochondria and bacteria. (6) To explore the feasibility of using group II introns as vectors for targeted gene disruption and site-specific DNA insertion in animal cells.

提出的研究涉及对II组内含子的蛋白质依赖性剪接和迁移率反应的持续生化基因研究。 II组内含子迁移率是由内含子编码的逆转录酶介导的，这些逆转录酶也起着成熟酶的作用，以促进内含子的剪接。 在当前赠款期间，我们阐明了蛋白质依赖性剪接和迁移率反应的一般性质，并开发了进一步研究反应的工具。 首先，我们表明，II组内含子迁移率（归纳）是通过靶标-DNA的逆转录机制进行的，涉及一种新型内含子编码的DNA核酸内切酶，该机制是一种含有内含子编码的蛋白质和切除的内含子RNA的RNP复合物。 在一种机制中，RNP复合物中的内含子RNA直接剪接到DNA靶位点，然后由内含子编码的蛋白反转录。 其次，我们表明确定内含子插入位点的II组内核酸内核酸内切酶，同时使用其RNA和蛋白质亚基来识别其DNA靶位点中的特定序列。 在内含子RNA的情况下，这涉及DNA靶位点的13-NT区域与内含子RNA中的序列之间的碱基配对，因此，可以简单地通过修改内含子RNA来改变内核酸酶的特异性。 这些发现增加了II组内含子可以用作靶向基因破坏的工具，并在DNA基因组中所需的位点插入遗传信息。 最后，我们开发了一个有效的大肠杆菌表达系统，用于乳酸乳酸菌II内含子L1.LTRB。 这项工作的最新扩展包括开发用于研究大肠杆菌中乳球菌内含子的恢复和剪接的遗传系统，以及使用纯化成分研究剪接和迁移率反应的方法。 具体目的是：（1）继续对成熟酶II组内含子剪接反应的生化分析。 （2）确定II组内含子剪接中涉及的其他蛋白质因子。 （3）分析内含子编码的DNA核酸内切酶活性的反应机理和DNA靶位点相互作用。 （4）支持协作以确定II组内含子编码蛋白的X射线晶体结构及其与内含子RNA和RT底物的复合物。 （5）继续在酵母线粒体和细菌的II组内含子迁移机制上进行合作。 （6）探索使用II组内含子作为靶向基因破坏和动物细胞中位点特异性DNA插入的载体的可行性。