INTRON DYNAMICS IN THE PHAGE/E COLI SYSTEM

在系统发育上分布广泛的第一组和第二组内含子是 动态的原创元素。它们通过不同的自我催化的RNA- 基于机制，而许多也能够插入到DNA中 通过不同的移动路径。对于每一类内含子，迁移性 其特点是采用“归位”的形式，即内含子 通过产生的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。而对于第一组归巢， 重组事件严格以DNA为基础，第二组归巢涉及 在模板和裂解酶水平上的RNA 机动性。这项工作的总体目标是利用原核基因 同时研究DNA和RNA内含子的系统 重新安排。 在过去的资助期间，我们在确定 基因工程中第一类内含子归位的结构和功能要求 T4噬菌体系统，并确定该过程由多个 重组途径。此外，我们还增进了对 通过鉴定E.Coli蛋白质进行蛋白质辅助的基于RNA的反应 在体外表现出RNA伴侣活性的物质。我们还获得了 支持移动基团I如何内含子的雌雄同体的证据 通过将内切酶基因入侵到编码 自剪接内含子。 在下一个资助期，我们建议延长界定 第一组流动过程。我们还将继续我们的工作 通过对StpA、E. 具有RNA伴侣活性的大肠杆菌蛋白，并通过分析 脉孢子菌剪接效应子CyT-18在大肠杆菌中的表达。另外，我们希望 为了将我们的工作扩展到研究细菌II组内含子归位，a 依赖于RNA剪接和DNA重组的过程。 因此，通过利用真细菌遗传系统并将 遗传分析、生化表征和 结构研究，我们建议增进我们对内含子- 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.