Transcription termination and antitermination in E.coli

描述(由申请人提供) 转录是所有细胞过程的关键，负责转录的RNA聚合酶(RNAP)是不同微生物病原体吸引的药物靶点。在特定的时间，大多数RNAP分子都参与了mRNA的合成和快速翻转，这涉及到转录的两个关键步骤，即延伸和终止。因此，理想的药物靶点应该是参与这两种模式的DNA结合的RNAP分子，而不是它们在胞浆中的游离形式。该项目的长期目标是了解转录过程中延长、终止以及抗终止步骤的机制方面，以便在未来有可能进行合理的药物设计。主要的焦点将是阐明RNAP的活性位点动力学和在这些步骤中复杂的蛋白质-DNA-RNA相互作用。噬菌体N-介导的抗终止系统，包括E.Coli RNAP，是研究转录延伸复合体中蛋白质-DNA-RNA相互作用以及了解终止/反终止过程的机制的理想系统。体内研究表明，志贺毒素噬菌体H19B需要噬菌体因子N和宿主因子NusA来修饰延长复合体，实现抗终止。因此，这种抗终止复合体在生化和结构研究方面要简单得多。通过突变、Fe-Babe裂解和荧光光谱来表征这种修饰延伸复合体中的相互作用。基于Taq RNA聚合酶和酵母RNA Pol II晶体结构的同源建模，结合上述实验获得的数据，将获得RNA聚合酶上N结合表面的三维定位。在平行研究中，将利用化学切割、足迹印迹、交联和荧光光谱等方法，通过实验研究RNAP对不同DNA序列、新生RNA结构和反式因子(如N蛋白等)的活性部位动力学。计算方法，如分子动力学模拟，也将被用来预测活性部位周围的结构域移动，这将被用来设计特定结构域的突变，并通过抑制遗传学找到其相互作用的伙伴。对活性部位动力学的了解将为未来合理的药物设计奠定基础。

DESCRIPTION (provided by applicant) Transcription is key to all the cellular processes and RNA polymerase (RNAP), the enzyme responsible for transcription, is an attractive drug target in different microbial pathogens. At a given time most of the RNAP molecules are engaged in mRNA synthesis and rapid turn over, which involves two crucial steps in transcription, namely elongation and termination. So, ideally drug target should be the DNA-bound RNAP molecules engaged in these two modes, rather than their free form in cytosol. Long term goal of this project is to understand the mechanistic aspects of elongation, termination and as well as antitermination steps of the transcription process, so that a rational drug designing will be possible in future. Major focus will be to elucidate the active site dynamics of RNAP and intricate protein-DNA-RNA interactions during these steps. N-mediated antitermination system from lamdoid phage, which involves E.Coli RNAP, is an ideal system to study the protein-DNA-RNA interactions in the transcription elongation complex and as well as to understand the mechanism of termination/antitermination processes. In vivo studies indicate that shiga-toxin bearing lamdoid phage, H19B, requires a phage factor N and the host factor NusA to modify the elongation complex and achieve antitermination. Therefore, this antitermination complex is much simpler for biochemical and structural studies. Interactions in this modified elongation complex will be characterized by mutagenesis, Fe-BABE cleavage and fluorescence spectroscopy. 3D localization of N-binding surface on RNA polymerase will be obtained from homology modeling based on Taq RNA polymerase and Yeast RNA Pol II crystal structures together with the data obtained from the experiments stated above. In parallel studies, the active-site dynamics of RNAP in response to different DNA sequences, nascent RNA structure and trans factors (like N protein etc.), will be studied experimentally by using, chemical cleavage, foot printing, cross linking, and fluorescence spectroscopy. Computational methods, such as molecular dynamics simulations will also be used to predict the domain movement around the active site, which will be used to design mutations in specific domains and find its interacting partners by suppressor genetics. Understanding of the active site dynamics will lay the foundation for rational drug design in future.