Structure/Function of Transcription Complex RNA Hairpins

描述(申请者提供)：本项目的远景目标是 定义转录复合体中调节暂停和转录的相互作用 用RNA聚合酶终止。新生的RNA发夹是重要的调控因素 细菌中的信号，其中停顿和终止是 基因调控机制。暂停和提前终止也会影响 在哺乳动物细胞和病毒中的基因表达，特别是与 癌症的发展和艾滋病病毒HIV-1的生长。 细菌和真核生物，专门的调节蛋白改变 转录复合体，使其抵抗停顿和终止。虽然 在理解暂停、终止和 控制这些事件的调节蛋白，两种不同的模型 仍然是可能的。在一种被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。在另一种情况下，这些信号和蛋白质主要影响 相对刚性的RNA聚合酶在RNA和DNA链上的易位 (刚体模型)。大肠杆菌RNA聚合酶暂停和终止 它们由NusA、NusG和RfaH蛋白调节，并由人RNA暂停 聚合酶II已被开发为模型系统。 将结合使用生化、遗传和生物物理方法。 区分转录的变构模型和刚体模型 规则，并描述暂停、终止和 控制它们的调节蛋白。具体目标将是(一) 鉴定RNA聚合酶的翻盖末端螺旋与RNA、NusA、 和Sigma-70，并测试这些相互作用如何影响活性 站点；(Ii)确定RNA3在暂停和结束时的位置 未暂停的转录延伸复合体；(Iii)测定动力学 延长、停顿和终止的机制；(Iv)MAP相互作用 RNA聚合酶与停顿和终止子发夹之间的关系；以及(V)确定 RfaH和NusG与RNA聚合酶相互作用的位点及其机制 它们控制着文字记录的延长。

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.