Molecular mechanism of transcription termination factor Rho as a RNA/DNA helicase

大肠杆菌rho转录终止蛋白是一种六美酶，它相信通过ATP-依赖的标记物中的RNA-DNA杂交来发挥作用。为了进一步简化Rho的三分子机制，我们专注于其结构相似性，使F-D21-D2-ATPase和处理以下两条研究线. 1）Rho ATPase反应的前稳态动力学：也是rho是一个同源的examer，在文学中的一些研究，认为六聚氰胺的六个子单位不是功能上唯一的。Equilibrium Nucleotide绑定实验表明，rho六examer上有3-4密集和2-3弱ATP绑定站点。为了了解rho六边形结合位点的两个类别的作用，我们已经测量了ATP羟基化和rho蛋白分离的动力学。结果表明，rho六聚氰胺有3个非催化站点，不能在快速ATP酶转换中参与，因此rho必须是催化站点上较弱的ATP绑定站点。2）Rho六聚氰胺环的三个局部重建从电子显微图像中：我们在一个20-A决议中产生了三个维度重建，并将tRNA分子绑定到主RNA-绑定站点到环的外部。N终端域的rho适合我们重建的原子结构，并且与RNA-绑定在环外的RNA-绑定中，在F-D21-类似D2的ATPase核心域的顶部转向位置。在这些观测的基础上，我们提出了一个Rho函数稳定四个基本步骤的模型：a） the binding of the rut site on a nascent transcript to the outside of the ring; b）the passage of the transcript into the central channel of the ring; c）the activation of the coupled ATPase-translocation within the central channel; and d）the linear translocation of rho along the transcript towards the polymerase。

The E coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid in an ATP-dependent manner. To further elucidate themolecular mechanism of Rho, we have focused on its structural similarity to FィイD21ィエD2-ATPase and conducted the following two lines of studies.1)Pre-steadystate kinetics of Rho ATPas reaction : Although rho is a homohexamer, several studies in the literature suggest that the six subunits of the hexamer are not functionally identical. Equilibrium nucleotide binding experiments have shown that there are 3-4 tight and 2-3 weak ATP binding sites on the rho hexamer. To understand the role of the two classes of nucleotide binding sites on the rho hexamer, we have measured the kinetics of ATP hydrolysis and nucleotide dissociation from the rho protein. The results indicate that the rho hexamer has 3 noncatalytic sites that do not participate in the fast ATPase turnover, and thus the weak ATP binding sites on rho must be the catalytic sites.2)Three-dimentional reconstruction of the Rho hexamer ring from electron microscopic images : We have generated a three -dimensional reconstruction of rho at a 20-A resolution, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring . An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring, which in turn sits on the top of the FィイD21ィエD2-like ATPase core domain.On the basis of these observations, we propose a model for the Rho function consisting of four elementary steps : a) the binding of the rut site on a nascent transcript to the outside of the ring; b)the passage of the transcript into the central channel of the ring; c)the activation of the coupled ATPase-translocation within the central channel; and d)the linear translocation of rho along the transcript towards the polymerase.