MECHANISM OF E. COLI TERMINATION FACTOR RHO

mRNA产生水平上的基因表达在转录起始和终止时都受到调控。终止因子Rho是一种从大肠杆菌转录复合体中释放新合成RNA的同六聚体蛋白。Rhos被认为是通过以atp为燃料的5‘->3’沿着新生RNA移动而起作用。这种旅行是通过Rho的RNA依赖性atp酶活性与RNA结合和释放的协调来实现的。当Rho到达转录RNA聚合酶时，它的持续旅行解开了转录泡的RNA- dna螺旋，从而破坏了三元转录复合物。这项工作的目标是了解Rho沿着RNA定向行进的机制。一种由真菌开发的抗生素，双环霉素，通过Rho抑制杀死革兰氏阴性菌。Rho在水解ATP时沿着RNA滚动的模型预测，环状排列的Rho亚基中的ATO水解将是连续的，并且顺序固定。这一预测将通过使用快速混合/化学淬火技术来检验预稳态ATP水解动力学来验证。动力学机制的其他特征也将被描述，包括配体结合顺序的确定。Rho的活性位点之间的催化协同性的证据将被追求和对协同性的要求进行表征。滚动模型还预测，当RNA与Rho结合时，一个未结合的5'区是Rho进行ATP水解的信号。这一预测将通过创建Rho的结合信号RNA分子多于或少于通常数量的情况来验证。每一种情况都预示着Rho对ATP水解模式的改变。这种模式将使用快速混合/化学淬火技术和核苷酸结合与同位素分配实验的组合来监测。在RNA结合相互作用中具有特征缺陷的Rho的变体形式将用于阐明Rho上的两种类型的RNA结合位点中的哪一种与RNA信号相互作用以进行ATP水解。提出的工作将测试和表征Rho沿RNA的ATP依赖定向行进的滚动模型，这是其转录终止活性的关键。该结果将更好地理解ATP水解的能量是如何转化为运动的。

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.