CAREER: Structural Basis of Transcription Termination in Thermophilic Eubacteria and Hypertext Curriculum for Advanced Undergraduate Biology Organized by Function and System

9734466 Friedman该项目旨在开始阐明转录终止的结构基础。转录终止似乎是各种因子之间以及它们与RNA聚合酶之间的多重弱相互作用的结果。已经确定的两种蛋白质是nusG和rho，它们对正确的转录终止至关重要。众所周知，大肠杆菌rho是一种解旋酶，可以在rho依赖的终止位点将RNA转录物从转录泡中解旋出来，而nusG则是rho和RNA聚合酶之间的接头。来自海苔的nusG同源物已经被证明很容易被蛋白水解成两个片段。两个碎片的结构将由x射线晶体学确定。同时，将利用现有的克隆构建海洋T. rho蛋白的表达系统。该蛋白的结晶实验也将进行，包括完整蛋白和蛋白水解裂解分离rna结合域。最后，将研究海洋T. nusG和rho蛋白之间的潜在相互作用，以确定它们是否也表现出与大肠杆菌中相同的相互作用。如果观察到这种相互作用，将启动两种蛋白质复合物的结晶试验。在CAREER计划的教育方面，将开发一套新的基于超文本的课程材料。这些材料将被设计成以“垂直”方向呈现生物学，重点关注生物功能或器官系统中不同层次组织之间的联系。课程材料的垂直组织应该有助于向本科生传达分子结构如何成为生物过程的基础。它还应该为高级生物学本科生提供另一种看待生物知识的视角，从而巩固他们在入门课程中所学到的知识。这种研究和教育项目的结合将有助于进一步将结构生物学融入生物学的主流。研究部分将为结构研究带来一个新的生物研究领域，而教育部分将有助于将我们现有的结构生物学知识整合到本科课程中。

9734466 Friedman The project is designed to begin to elucidate the structural basis of transcription termination. Transcriptional termination appears to result from multiple weak interactions among various factors and between them and the RNA polymerase. Two proteins that have been identified as being critical for proper transcription termination are nusG and rho. E. coli rho is known to be a helicase that unwinds the RNA transcript from the transcription bubble at rho-dependent termination sites, while nusG acts as an adaptor between rho and the RNA polymerase. The nusG homolog from T. maritima has been demonstrated to be readily cleaved by proteolysis into two fragments. The structures of both fragments will be determined by x-ray crystallography. At the same time, expression systems for the T. maritima rho protein will be constructed from presently available clones. Crystallization experiments of this protein will also be conducted, both with the intact protein and protein proteolytically cleaved to separate the RNA-binding domain. Finally, potential interactions between the T. maritima nusG and rho proteins will be investigated to determine if they also show the same interaction which is seen in their E. coli counterparts. If this interaction is seen, crystallization trials on a complex of the two proteins will be initiated. On the education side of this CAREER proposal, a new set of hypertext-based curriculum materials will be developed. These materials will be designed to present biology in a "vertical" direction, focusing on the connections between different levels of organization within a biological function or organ system. The vertical organization of the curriculum material should help communicate to under-graduate students how molecular structure underlies biological processes. It should also provide upper-level biology undergraduates with an alternative view of their biological knowledge, thereby solidifying what they have been taught in their introductory classes. This combination of research and educational projects will help further integrate structural biology into the mainstream of biology. The research component will bring a new area of biological research under structural investigation, while the education component will help integrate our present knowledge of structural biology into the undergraduate curriculum.