Mechanism of Transcription Termination Protein Rho

大肠杆菌转录终止蛋白Rho利用依赖于RNA的ATPase活性来影响暂停转录复合体的转录释放。PI正在利用酶学、生化和分子生物学技术研究Rho ATPase反应的分子机制，以了解其在转录终止中的作用。所获得的知识将被证明对更广泛的RNA-蛋白质相互作用问题和对能量转导ATPase的理解是有用的。在本提案中，继续对Rho ATPase活性的研究，并开始研究Rho与RNA的相互作用。Rho被认为利用ATP水解的能量相对于转录复合体的RNA链移动。Rho与这种RNA结合，并从RNA聚合酶和DNA模板中释放它。这种运动的假设机制是依赖于ATP水解的新生RNA的结合和释放。根据早期关于Rho-ATP相互作用的工作，预测RNA与Rho ATP复合体的结合应该比与Rho ADP.PI复合体更紧密，并设计了一个实验来验证这一预测。为了测试Rho是否利用ATP水解的能量相对于RNA旅行，Stitt博士和他的同事开发了一种系统来测量每一条RNA链释放的ATP水解量。该系统用于正常的Rho依赖释放；它也将用于在Rho结合位点和转录本末端之间插入的系统中。如果Rho使用ATP能量沿着RNA旅行，它应该在系统中使用更多的ATP，在那里它必须走得更远。Rho上与RNA相互作用的位置仅被模糊地定位；RNA交联实验将被用来更好地定义它们。Rho显示需要RNA激活其ATPase活性，尽管DNA聚(DC)也与Rho结合作为RNA聚(C)。确定序列的混合RNA-DNA寡核苷酸将被用来确定RNA上2‘OH对Rho ATPase激活至关重要的位置。

Escherichia coli transcription termination protein rho employs an RNA-dependent ATPase activity to effect transcript release from paused transcription complexes. The PI is studying the molecular mechanism of the rho ATPase reaction using enzymological, biochemical, and molecular biological techniques, in order to understand its role in transcription termination . The knowledge gained will prove useful to the broader question of RNA-protein interactions and to the understanding of energy-transducing ATPases. In the present proposal, studies of rho ATPase activity are continued, and an investigation of rho-RNA interactions is begun. RHO is thought to use the energy of ATP hydrolysis to move with respect to the RNA chain of transcription complexes. Rho binds to this RNA and releases it from RNA polymerase and the DNA template. The postulated mechanism for such movement is the ATP hydrolysis- dependent binding and release of the nascent RNA. From earlier work on rho-ATP interactions it is predicted that RNA should bind to rho ATP complexes much more tightly than to rho ADP.Pi complexes, and an experiment had been designed to test this prediction. To test whether rho uses the energy of ATP hydrolysis to travel with respect to RNA, Dr. Stitt and co- workers have developed a system to measure the amount of ATP hydrolyzed per RNA chain released. This system is being used in a normal rho-dependent release; it will also be employed in a system in which an insert has been made between the rho binding site and the end of the transcript. If rho used ATP energy to travel along RNA, it should use more ATP in the system where it has to travel further. The sites on rho of its interaction with RNA have only vaguely been localized; RNA crosslinking experiments will be used to better define them. Rho shows a requirement for RNA to activate its ATPase activity, although the DNA poly(dC) binds as well to rho as the RNA poly(C). Mixed RNA-DNA oligonucleotides of defined sequence will be used to identify the locations on RNA where the 2'OH is critical for rho ATPase activation.