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.

拟议的研究涉及对第二组内含子的蛋白质依赖性剪接和移动反应的持续生化遗传学研究。 II组内含子移动性由内含子编码的逆转录酶介导，该逆转录酶也充当成熟酶以促进内含子的剪接。 在当前的资助期内，我们阐明了蛋白质依赖性剪接和迁移反应的一般性质，并开发了进一步研究这些反应的工具。 首先，我们证明了 II 组内含子迁移（归巢）是通过靶 DNA 引发的逆转录机制发生的，该机制涉及一种新型内含子编码的 DNA 核酸内切酶，该酶是一种包含内含子编码蛋白和切除的内含子 RNA 的 RNP 复合物。 在一种机制中，RNP 复合物中的内含子 RNA 直接反向剪接到 DNA 靶位点，然后由内含子编码的蛋白质进行逆转录。 其次，我们表明，确定内含子插入位点的 II 组内含子核酸内切酶使用其 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，可以简单地通过修饰内含子 RNA 来改变核酸内切酶的特异性。 这些发现提出了第二组内含子可能被用作靶向基因破坏和将遗传信息插入 DNA 基因组中所需位点的工具的可能性。 最后，我们开发了一种针对乳酸乳球菌 II 组内含子 L1.LtrB 的高效大肠杆菌表达系统。 最近的工作扩展包括开发用于研究大肠杆菌中乳球菌内含子的逆向归巢和剪接的遗传系统，以及使用纯化成分研究剪接和迁移反应的方法。 具体目标是： (1) 继续对成熟酶依赖性第二组内含子剪接反应进行生化分析。 (2) 鉴定参与 II 组内含子剪接的其他蛋白质因子。 (3)分析内含子编码的DNA核酸内切酶活性的反应机制和DNA靶位点相互作用。 (4) 支持合作确定 II 族内含子编码蛋白及其与内含子 RNA 和 RT 底物的复合物的 X 射线晶体结构。 (5) 继续在酵母线粒体和细菌中II组内含子迁移机制方面开展合作。 (6) 探讨使用II族内含子作为载体在动物细胞中进行靶向基因破坏和位点特异性DNA插入的可行性。