STRUCTURE/FUNCTION OF TRANSCRIPTION COMPLEX RNA HAIRPINS

描述：Landick博士提交了一份继续他的研究的提案。 对E. coli RNA聚合酶。 主要的题目是他的领导人的停顿部位。 在过去的项目中 在此期间，他取得了令人印象深刻的进展，这在进度报告中得到了证明。 以及大量的出版物（19篇在同行评审期刊上，3篇 已提交，2份）。 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 以及停顿期间的变化，并进一步定义他的停顿 信号了 这一庞大的工作机构作出了重要的概念贡献 我们对转录过程的理解。 例如，十字架 将所述暂停发夹连接至所述亚基的特定区域，并且将所述暂停发夹连接至所述亚基的特定区域。 RNA聚合酶构象的同时明显变化 相信暂停至少部分是由这种 交互. 兰迪克博士和他的同事和合作者的工作 还产生了一种新的重要方法， 可以监测单个RNA聚合酶分子。 现在就是这样 可以确定从以下获得的平均结果的分量： 传统的分子生物学实验 对于下一个项目期间，Landick博士提出了一系列逻辑 这项工作的延伸。 该架构的工作将扩展到 新生转录物3'端的定位和停顿的作用 发夹将进一步调查。 此外，还将努力 RNA聚合酶的最佳图谱位置和构象 在DNA模板上移位并暂停。 希望这将 有助于区分蠕动模型和滑动模型。 一个非常重要 下一阶段的任务是测试NTP浓度是否有任何影响 暂停和终止转录期间的事件。 先前 所有的实验都是用非常低的非生理性GTP完成的 浓度（5 μ M），以减缓反应。 转录 在更高GTP浓度下的实验将通过视频进行监控 单个聚合酶的显微镜以及停流动力学。 此外，转录因子，如NusA和NusG的作用将 因为这些因素在早期的实验中被忽略了。 遗传学将被用来完善结构的定义。 RNA聚合酶的元件在暂停和 终止，并将试图评估暂停在 新生RNA转录物的折叠。 最后，遗传学也将成为 用于评估衰减过程中暂停的体内作用 在他的领导者。

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.