Mechanism of Transcription Termination Protein Rho

大肠杆菌转录终止蛋白rho采用 RNA依赖性ATP酶活性影响转录物释放 从暂停的转录复合体中分离出来 PI正在研究 rho ATP酶反应的分子机制， 酶学、生物化学和分子生物学 技术，以了解其在转录中的作用 终止 所获得的知识将证明对 更广泛的RNA-蛋白质相互作用的问题， 对能量转换ATP酶的理解。 本 建议，rho ATP酶活性的研究继续进行， 开始了rho-RNA相互作用的研究。 RHO被认为 利用ATP水解的能量， 转录复合物的RNA链。 Rho与这种RNA结合 从RNA聚合酶和DNA模板中释放出来。 的 这种运动的假定机制是ATP水解- 依赖于新生RNA的结合和释放。 从早期 研究rho-ATP相互作用，预测RNA应该 与rho ATP复合物的结合比与rho.ADP.Pi的结合更紧密 复合物，并设计了一个实验来测试这一点 预测. 为了测试rho是否利用ATP的能量 关于RNA的水解旅行，Stitt博士和他的同事 工作人员开发了一种测量ATP含量的系统， 每释放一条RNA链水解。该系统正在被用于 正常的rho依赖性释放;它也将用于 在rho绑定之间插入的系统 网站和成绩单的结尾。 如果rho使用ATP能量 沿着RNA，它应该在系统中使用更多的ATP， 必须走得更远。 它的相互作用在ρ上的位置 与RNA只有模糊地定位; RNA交联 实验将被用来更好地定义它们。 Rho显示a 需要RNA激活其ATP酶活性，尽管 DNA策略（dC）与RNA-DNA一样与rho结合 确定序列的寡核苷酸将用于鉴定 RNA上2 'OH对rho ATP酶至关重要的位置 activation.

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 use to better define them. Rho shows a requirement for RNA to activate its ATPase activity, although the DNA policy (dC) binds as well to rho as the 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.