INTRON DYNAMICS IN THE PHAGE/E COLI SYSTEM

遗传学上广泛分布的I组和II组内含子是 动态遗传因素 它们通过不同的自催化RNA进行剪接- 基于机制，而许多也能够插入到DNA 通过不同的移动路径。 对于每类内含子， 典型地采取“归巢”的形式， 通过双链断裂转移到无内含子的DNA等位基因， 由内含子编码的核酸内切酶。 而对于I组归航， 重组事件是严格基于DNA的，II组归巢涉及 RNA在模板和切割酶的水平上， 迁移率 这项工作的总体目标是利用原核遗传 研究基于DNA和基于RNA的内含子的系统 重新安排 在过去的资助期间，我们在确定 I组内含子归巢的结构和功能要求 T4噬菌体系统，并确定该过程发生的多个 重组途径 此外，我们进一步了解到， 通过鉴定E.杆菌蛋白 其在体外表现出RNA伴侣活性。 我们也得到 支持移动的I组内含子 进化，通过核酸内切酶基因侵入编码 自我剪接内含子。 在下一个资助期内，我们建议延长研究， 第一组流动程序。 我们还将继续努力， 蛋白质辅助剪接，通过探讨StpA的机制，一个E. 大肠杆菌蛋白的RNA伴侣活性，并通过分析 链孢菌剪接效应子CYT-18在大肠杆菌中的表达。杆菌 此外，我们希望 为了将我们的工作扩展到研究细菌II组内含子归巢， 这一过程依赖于RNA剪接和DNA重组。 因此，通过利用真细菌遗传系统并结合 遗传分析、生化表征和 结构研究，我们建议推进我们的理解内含子- 相关的核酸动力学。

The phylogenetically widespread group I and group II introns are dynamic gentic elements. They splice by different self-catalyzed RNA- based mechanisms, while many are also capable of insertion into DNA through distinct mobility pathways. For each calss of intron, mobility characteristically takes the form of 'homing', whereby the intron transfers to an intronless DNA allele via a double-strand break created by an intron-encoded endonuclease. Whereas for group I homing, recombination events are strictly DNA-based, group II homing involves RNA at levels of both the template and the cleavage enzyme for mobility. The overall goal of this work is to use prokaryotic genetic systems to study both DNA-based and RNA-based intron rearrangements. During the past funding period we made progress towards defining structural and functional requirements of group I intron homing in the T4 phage system and determined that the process occurs by multiple recombination pathways. Furthermore, we advanced our understanding of protein-assisted RNA-based reactions by identifying E. Coli proteins that exhibit RNA chaperone activity in vitro. We also obtained evidence that supports an gypothesis for how mobile group I introns evolved, by invasion of an endonuclease gene into DNA encoding the self-splicing intron. For the next funding period we propose to extend the studies defining the group I mobility process. We will also continue our work on protein-assisted splicing, by probing the mechanism of StpA, an E. Coli protein with RNA chaperone activity, and by analyzing the Neurospora splicing effector CYT-18 in E. coli. Additionally, we wish to extend our work to studying bacterial group II intron homing, a process dependent upon both RNA splicing and DNA recombination. Thus, by exploiting eubacterial genetic systems and combining the approaches of genetic analysis, biochemical characterization and structural study, we propose to advance our understanding of intron- related nucleic acid dynamics.