Mechanism of E. coli Transcription Termination Factor Rho

大肠杆菌的转录终止因子Rho从转录复合体中释放RNA。Rho是一种依赖于RNA的ATP酶，它利用ATP水解产生的能量沿着RNA移动5‘ - 3’。这种旅行可能构成Rho的RNA- dna解旋酶功能的基础，该功能被认为参与转录复合物的破坏和RNA的释放。长期目标是在分子水平上理解，是如何工作的。基于晶体结构和与其他蛋白质的比较，Rho的氨基酸在rna依赖的ATP水解中具有关键的活性位点功能。然而，对于许多催化位点的作用，有多个候选物。将进行定点诱变和所产生蛋白质的测试，以确定相关氨基酸并查明其功能。Rho构象变化的识别也将通过基于肽的氢/氘酰胺质子交换来分析。这些研究将揭示当底物、产物或过渡态类似物结合时，酰胺氢交换在Rho内发生变化的位置。研究结果将揭示Rho参与ATP水解和定向移动的构象变化，并将改善目前Rho- rna相互作用的不完整图景。结合这两种实验方法的结果将有助于更好地理解ATP水解与蛋白质构象变化的协调。蛋白质沿核酸的定向运动具有重要的基础意义和广泛的存在；这项工作将使我们有一个更全面的了解。在研究过程中对研究生和本科生进行培训，研究成果将在科学会议上发表，并在科学期刊上发表。

Transcription termination factor Rho of E. coli releases RNA from transcription complexes. Rho, an RNA-dependent ATPase, uses the energy from ATP hydrolysis to travel 5' - 3' along RNA. This travel may constitute the basis for the RNA-DNA helicase function of Rho, which is believed to be involved in disruption of transcription complexes and release of RNA. The long-term goal is to understand, at the molecular level, how Rho works. Amino acids of Rho that have critical active site functions in RNA-dependent ATP hydrolysis have been proposed based on crystal structures and comparisons with other proteins. There are, however, multiple candidates for many of the catalytic site roles. Site-directed mutagenesis and testing of the resultant proteins will be carried out both to identify the relevant amino acids and to pinpoint their functions. The identification of conformation changes in Rho will also be analyzed by peptide-based hydrogen/deuterium amide proton exchange. These studies will reveal the locations within Rho where amide hydrogen exchange changes when substrates, products, or transition state analogs bind. The results should reveal conformation changes that are involved in ATP hydrolysis and directional travel by Rho, and will enhance the present incomplete picture of Rho-RNA interactions. Combining the results of these two experimental approaches will allow better understanding of the coordination of ATP hydrolysis with protein conformation change. Directional protein travel along nucleic acids is of fundamental importance and wide occurrence; a more complete understanding will result from this work. Training will be provided to graduate and undergraduate students during the research, and results will be presented at scientific meetings and published in scientific journals.