Mechanism of Transcription Termination Protein Rho

大肠杆菌转录终止蛋白rho利用rna依赖的atp酶活性来影响转录物从暂停的转录复合物中释放。PI正在使用酶学，生化和分子生物学技术研究rho atp酶反应的分子机制，以了解其在转录终止中的作用。所获得的知识将证明对rna -蛋白质相互作用的更广泛问题和对能量转导atp酶的理解是有用的。在目前的建议中，继续研究rho atp酶活性，并开始研究rho- rna相互作用。RHO被认为是利用ATP水解的能量相对于转录复合物的RNA链移动。Rho与RNA结合并将其从RNA聚合酶和DNA模板中释放出来。这种运动的假设机制是ATP水解依赖于新生RNA的结合和释放。从早期关于rho-ATP相互作用的研究中可以预测，RNA与rho ATP复合物的结合要比与rho ADP的结合紧密得多。圆周率复合体，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水解的能量在RNA上移动，Stitt博士和同事开发了一个系统来测量每条释放的RNA链水解的ATP量。该系统被用于正常的rho依赖性释放；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。如果利用ATP能量沿着RNA移动，它就应该在系统中使用更多的ATP，这样它就必须走得更远。它与RNA相互作用的rho位点仅被模糊定位；RNA交联实验将用于更好地定义它们。Rho显示需要RNA来激活它的atp酶活性，尽管DNA多聚体（dC）和RNA多聚体(C)一样能与Rho结合。定义序列的混合RNA- dna寡核苷酸将用于鉴定RNA上的2'OH对rho atp酶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.