Transcription termination and antitermination in E.coli

大肠杆菌中的转录终止和抗终止

• 批准号: 6767789
• 负责人: RANJAN SEN
• 金额: $ 5.4万
• 依托单位: CTR FOR DNA FINGERPRINTING/DIAGNOSTICS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-18 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767789
• 关键词: DNA directed RNA polymerase; DNA footprinting; Escherichia coli; Mycobacterium tuberculosis; active sites; antisense nucleic acid; computer simulation; conformation; crosslink; fluorescence resonance energy transfer; fluorescence spectrometry; genetic transcription; messenger RNA; molecular dynamics; nucleic acid structure; protein protein interaction; transcription factor; transcription termination

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.

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中有吸引力的药物靶标。在给定的时间，大多数RNAP分子参与mRNA的合成和快速翻转，这涉及转录中的两个关键步骤，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RNAP分子，而不是它们在胞质溶胶中的游离形式。本项目的长期目标是了解转录过程中的延伸、终止和抗终止步骤的机制，以便将来能够进行合理的药物设计。主要重点将是阐明RNAP的活性位点动力学和复杂的蛋白质-DNA-RNA相互作用在这些步骤。来自lamdoid噬菌体的N-介导的抗终止系统涉及大肠杆菌RNAP，是研究转录延伸复合体中蛋白质-DNA-RNA相互作用以及理解终止/抗终止过程机制的理想系统。体内研究表明，志贺毒素轴承lamdoid噬菌体，H19 B，需要一个噬菌体因子N和宿主因子NusA修改的延伸复合物，并实现抗终止。因此，这种抗终止复合物对于生物化学和结构研究来说要简单得多。在这个修改后的延伸复合物的相互作用将通过诱变，Fe-BABE裂解和荧光光谱的特点。RNA聚合酶上的N-结合表面的3D定位将从基于Taq RNA聚合酶和酵母RNA Pol II晶体结构的同源建模以及从上述实验获得的数据获得。在平行的研究中，RNAP的活性位点动态响应不同的DNA序列，新生RNA结构和反式因子（如N蛋白等），将通过使用化学裂解、足迹、交联和荧光光谱进行实验研究。计算方法，如分子动力学模拟也将用于预测活性位点周围的结构域运动，这将用于设计特定结构域中的突变，并通过抑制遗传学找到其相互作用的伴侣。对活性中心动力学的了解将为今后合理的药物设计奠定基础。