GROUP II INTRON RNA SPLICING AND MOBILITY

拟议的研究涉及继续进行的生物化学遗传学研究的蛋白质依赖性剪接和流动性反应的第二组内含子。 II组内含子迁移率由内含子编码的逆转录酶介导，所述逆转录酶也作为成熟酶起作用以促进内含子的剪接。 在目前的资助期间，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反应的工具。 首先，我们表明，II组内含子迁移（归巢）发生通过靶DNA引发的逆转录机制，涉及一种新的内含子编码的DNA核酸内切酶，这是一个RNP复合物含有内含子编码的蛋白质和切除的内含子RNA。 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逆转录。 第二，我们表明，第二组内含子核酸内切酶，决定内含子插入的网站，使用它们的RNA和蛋白质亚基，以识别在其DNA靶位点的特定序列。 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来改变内切核酸酶的特异性。 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在DNA基因组中所需位点插入遗传信息的工具的可能性。 最后，我们开发了一个有效的E。乳酸乳球菌Ⅱ组内含子L1.LtrB的原核表达系统。 最近的扩展工作包括遗传系统的开发，用于研究大肠杆菌中乳球菌内含子的归巢和剪接。大肠杆菌中，以及使用纯化的组分研究剪接和迁移反应的方法。 具体目标是：（1）继续成熟酶依赖的II组内含子剪接反应的生化分析。 (2)确定参与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内切酶活性的反应机理和DNA靶位点相互作用。 (4)支持合作，以确定第二组内含子编码的蛋白质及其与内含子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第二组内含子移动机制方面的合作。 (6)探讨II类内含子作为动物细胞靶向基因破坏和定点DNA插入载体的可行性。

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.