STRUCTURE/FUNCTION OF TRANSCRIPTION COMPLEX RNA HAIRPINS

描述：兰迪克博士提交了一份提案，要求继续他的 研究大肠杆菌RNA聚合酶的转录暂停和终止。 主要的话题是《他的领袖》中的停顿地点。在过去的项目中 在此期间，正如进度报告所证明的那样，他取得了令人印象深刻的进展 以及大量出版物(19份在同行评议期刊上，3份在同行评议期刊上 评论，提交2篇)。在三个方面取得了实质性进展 区域：暂停所涉及的子单元上的功能域映射 和终止，阐明了转录复合体的结构 以及在停顿期间这方面的变化，以及对HIS停顿的进一步定义 信号。这一大堆工作在概念上做出了重要贡献 有助于我们对转录过程的理解。例如，十字架 将暂停的发夹连接到亚基的特定区域和 RNA聚合酶构象的同步明显变化 相信这样一个模型，即停顿至少部分是由这样的情况引起的 互动。兰迪克博士及其同事和合作者的工作 还产生了一种新的重要方法，通过这种方法转录 单个RNA聚合酶分子可以被监测。因此，现在就是这样 可以确定从以下公式获得的平均结果的分量 传统的分子生物学实验。 在接下来的项目期间，兰迪克博士提出了一系列符合逻辑的 这项工作的延伸。架构方面的工作将扩大到 初生成绩单3‘端的映射和停顿的作用 发夹将进行进一步调查。此外，还将尝试 RNA聚合酶在ITS过程中更好的MAP位置和构象 DNA模板上的移位和暂停。希望这将是 帮助区分英寸蠕虫模型和滑动模型。一个非常重要的 下一阶段的任务是测试NTP浓度是否有任何影响 关于转录暂停和终止过程中的事件。上一首 所有的实验都是在非常低和非生理的GTP下进行的 浓度(5微米)，以减缓反应。转录 更高GTP浓度的实验将通过视频进行监控 单个聚合酶的显微镜观察以及停流动力学。 此外，转录因子，如NusA和NusG的作用将 由于这些因素在以前的实验中已被省略，因此需要进行评估。 遗传学将被用来完善结构的定义 RNA聚合酶中发挥功能的元件，在暂停和 终止，并尝试评估暂停的可能作用 新生核糖核酸转录本的折叠。最后，遗传学也将是 用来评估停顿在体内对衰减过程的作用 在他的领袖中。

DESCRIPTION: Dr. Landick has submitted a proposal for continuation of his work on transcription pausing and termination of the E. coli RNA polymerase. The main topic is the pause site in the his leader. During the past project period he has made impressive progress as evidenced in the progress report and the large number of publications (19 in peer-reviewed journals, 3 reviews, and 2 submitted). Substantial progress has been made in three areas: mapping of functional domains on the subunits involved in pausing and termination, elucidated the architecture of the transcription complex and changes in this during pausing, and further definition of the his pause signal. This large body of work has made important conceptual contributions to our understanding of the transcription process. For example, the cross linking of the pausing hairpin to a specific region of the subunit and the simultaneous apparent change in the RNA polymerase conformation lends credence to the model that pausing is induced, at least in part, by such interactions. The work of Dr. Landick and his coworkers and collaborators has also generated a novel important method by which the transcription of individual RNA polymerase molecules can be monitored. Thus it is now possible to determine the components of the average results obtained from traditional molecular biology experiments. For the next project period Dr. Landick has proposed a series of logical extensions of this work. Work on the architecture will be expanded to mapping of the 3' end of the nascent transcript and the role of the pausing hairpin will be further investigated. Furthermore, it will be attempted to better map position and conformation of RNA polymerase during its translocation on the DNA template and pausing. It is hoped that this will help distinguish between inch-worm and sliding models. A very important task for the next period is to test if the NTP concentration has any effect on the events during pausing and termination of transcription. Previous experiments have all been done with a very low and unphysiological GTP concentration (5muM) in order to slow the reaction. Transcription experiments at higher GTP concentration will be monitored with video microscopy of individual polymerases as well as stop-flow kinetics. Furthermore, the role of transcription factors, such as NusA and NusG will be assessed, since these factors have been omitted from earlier experiments. Genetics will be employed to refine the definition of the structural elements of the RNA polymerase that play a functional role in pausing and termination and it will be attempted to assess possible roles of pausing in the folding of the nascent RNA transcript. Finally, genetics will also be employed to assess the in vivo role of pausing for the attenuation process in the his leader.