Transcription termination and antitermination in E.coli

大肠杆菌中的转录终止和抗终止

• 批准号: 6932965
• 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/6932965
• 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和RNA聚合酶（RNAP）（负责转录的酶）的关键，它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分子都从事mRNA合成和快速翻转，这涉及转录的两个关键步骤，即伸长和终止。因此，理想情况下，药物靶标应该是以这两种模式参与的DNA结合的RNAP分子，而不是其在细胞质中的自由形式。该项目的长期目标是了解转录过程的伸长，终止以及抗授权步骤的机理方面，以便将来可以进行理性的药物设计。主要重点是在这些步骤中阐明RNAP和复杂的蛋白质-DNA-RNA相互作用的主动位点动力学。涉及大肠杆菌RNAP的lamdoid Phage的N介导的抗授权系统是研究转录伸长复合物中蛋白DNA-RNA相互作用的理想系统，并了解终止/抗抑制过程的机理。体内研究表明，shiga-toxin轴承lamdoid噬菌体H19B需要噬菌体因子N和宿主因子NUSA来修饰伸长率并实现抗逆转录。因此，对于生化和结构研究，这种抗授权复合物要简单得多。这种修饰的伸长络合物中的相互作用的特征是诱变，fe子裂解和荧光光谱。 N结合表面在RNA聚合酶上的3D定位将从基于TAQ RNA聚合酶和酵母RNA POL II晶体结构的同源性建模以及从上述实验获得的数据获得。在平行研究中，将通过使用化学裂解，脚部印刷，交叉链接和荧光光谱法对RNAP的活性位点动力学（如新生的RNA结构和反式因子（例如N蛋白等））进行实验研究。计算方法（例如分子动力学模拟）也将用于预测活性位点周围的域运动，该域将用于在特定域中设计突变，并通过抑制器遗传学找到其相互作用的伴侣。对主动现场动态的理解将为未来的理性药物设计奠定基础。