Structure/Function of Transcription Complex RNA Hairpins

描述（由申请人提供）：本项目的长期目标是 定义转录复合物中调节暂停的相互作用， RNA聚合酶终止。新生RNA发夹是重要的调控因子 细菌中的信号，其中暂停和终止是 基因调控机制。暂停和过早终止也会影响 基因在哺乳动物细胞和病毒中的表达，特别是涉及 癌症的发展和艾滋病病毒HIV- 1的生长。无论是 细菌和真核生物，专门的调节蛋白质修饰 转录复合物，使其耐暂停和终止。虽然 在理解暂停、终止和 控制这些事件的调节蛋白， 仍然是可能的。在一种称为变构模型的观点中，暂停信号， 终止信号和调节蛋白主要影响构象 RNA聚合酶。另一方面，这些信号和蛋白质主要影响 RNA和DNA链上相对刚性的RNA聚合酶的移位 (the刚体模型）。暂停和终止由E. coli RNA聚合酶和 它们受NusA、NusG和RfaH蛋白的调节，并受人RNA的暂停 聚合酶II已被开发为模型系统。 将采用生物化学、遗传学和生物物理学方法相结合的方法 为了区分转录的变构和刚体模型， 调节，并表征暂停，终止和 控制它们的调节蛋白。具体目标是：（一） 表征RNA聚合酶的瓣尖螺旋与RNA，NusA， 和sigma-70，并测试这些相互作用如何影响活性 （ii）确定RNA 3在暂停末端附近的位置， 非暂停转录延伸复合物;（iii）确定动力学 延长、暂停和终止的机制;（iv）地图相互作用 RNA聚合酶与暂停和终止子发夹之间的相互作用;和（v）确定RNA聚合酶与暂停和终止子发夹之间的相互作用。 RfaH和NusG与RNA聚合酶相互作用的位点以及 它们调节转录延长。

DESCRIPTION (provided by applicant): The long-range goal of this project is to define the interactions in the transcription complex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV- 1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Although significant progress has been made in understanding pausing, termination, and the regulatory proteins that control these events, two alternative models remain possible. In one view, called the allosteric model, pause signals, termination signals, and regulatory proteins primarily affect the conformation of RNA polymerase. In the other, these signals and proteins primarily affect translocation of a relatively rigid RNA polymerase on the RNA and DNA chains (the rigid-body model). Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems. A combination of biochemical, genetic, and biophysical approaches will be used to distinguish the allosteric and rigid-body models of transcriptional regulation, and to characterize the mechanisms of pausing, termination, and regulatory proteins that control them. Specific aims will be to (i) characterize interactions of RNA polymerase's flap-tip helix with RNA, NusA, and sigma-70, and test how these interactions affect catalysis in the active site; (ii) determine the location of the RNA 3 about end in paused and nonpaused transcription elongation complexes; (iii) determine the kinetic mechanisms of elongation, pausing, and termination; (iv) map interactions between RNA polymerase and pause and terminator hairpins; and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation.