MECHANISM OF E. COLI TERMINATION FACTOR RHO

大肠杆菌终止因子 RHO 的作用机制

• 批准号: 6387040
• 负责人: BARBARA L. STITT
• 金额: $ 15.75万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387040
• 关键词: Escherichia coli; RNA binding protein; active sites; adenosinetriphosphatase; bacterial RNA; bacterial proteins; enzyme mechanism; enzyme model; enzyme substrate complex; protein engineering; transcription factor; transcription termination

Gene expression at the level of mRNA production is regulated both at the initiation and termination of transcription. Termination factor Rho is a homohexameric protein that releases newly synthesized RNA from Escherichia coli transcription complexes. Rhos is thought to act through ATP-fueled, 5'->3' travel along nascent RNA. This travel is achieved by coordination of the RNA-dependent ATPase activity of Rho with RNA binding and release. Upon the arrival of Rho at the transcribing RNA polymerase, its continued travel unwinds the RNA-DNA helix of the transcription bubble, which disrupts the ternary transcription complex. The goal of the proposed work is the understanding the mechanism of directional travel by Rho along RNA. A fungal-developed anti-biotic, bicyclomycin, kills Gram-negative bacteria through Rho inhibition. A model in which Rho rolls along RNA as it hydrolyzes ATP predicts that ATO hydrolysis in the circularly arrayed Rho subunits will be sequential and in a fixed order. This prediction will be tested by using rapid mix/chemical quench techniques to examine pre-steady-state ATP hydrolysis kinetics. Other features of the kinetic mechanism will also be characterized, including determination of ligand binding order. Evidence for catalytic cooperativity among the active sites of Rho will be pursued and the requirements for cooperativity characterized. The rolling model also predicts that when RNA is bound to Rho, an unbound 5' region is the signal to Rho for ATP hydrolysis. This prediction will be tested by creating situations in which Rho has more or fewer than the usual number of signal RNA molecules bound. Each of these situations predicts changes in the pattern of ATP hydrolysis by Rho. This pattern will be monitored using rapid mix/chemical quench techniques and a combination of nucleotide binding with isotope partitioning experiments. Variant forms of Rho with characterized defects in RNA binding interactions will be used to clarify which of the two types of RNA binding sites on Rho interacts with the RNA signal for ATP hydrolysis. The proposed work will test and characterize the rolling model for ATP- dependent directional travel by Rho along RNA, which is the key to its transcription termination activity. The results will provide a better understanding of how the energy of ATP hydrolysis can be transduced to movement.

在转录的起始和终止阶段，基因表达在信使核糖核酸的产生水平上都受到调控。终止因子Rho是一种从大肠杆菌转录复合体中释放新合成的RNA的同源六聚体蛋白。Rhos被认为是通过以ATP为燃料的5‘-gt；3’沿着新生的RNA旅行而起作用的。这种旅行是通过协调Rho的RNA依赖的ATPase活性与RNA的结合和释放来实现的。当Rho到达转录RNA聚合酶时，它的持续旅行解开了转录泡泡的RNA-DNA螺旋，从而破坏了三元转录复合体。这项拟议工作的目标是理解Rho沿RNA定向旅行的机制。一种真菌开发的抗菌素双环素，通过抑制Rho杀死革兰氏阴性细菌。当Rho水解ATP时，Rho沿着RNA滚动的模型预测，环状排列的Rho亚基中的ATO水解会是有序的，并以固定的顺序进行。这一预测将通过使用快速混合/化学猝灭技术来检验稳定前的ATP水解动力学。动力学机制的其他特征也将被表征，包括配体结合顺序的确定。将寻求Rho活性中心之间催化协作性的证据，并表征协作性的要求。滚动模型还预测，当RNA与Rho结合时，未结合的5‘区是Rho进行ATP水解的信号。这一预测将通过创造条件来验证，在这种情况下，Rho结合的信号RNA分子比通常的多或少。这些情况中的每一种都预示着Rho对ATP的水解模式发生了变化。这种模式将使用快速混合/化学猝灭技术以及核苷酸结合和同位素分配实验相结合的方法进行监测。具有RNA结合作用缺陷的Rho变体将被用来阐明Rho上两种类型的RNA结合位点中的哪一种与ATP水解的RNA信号相互作用。这项拟议的工作将测试和表征Rho沿RNA依赖于ATP的定向旅行的滚动模型，这是其转录终止活性的关键。这些结果将使我们更好地理解ATP水解的能量是如何转化为运动的。