Transcription termination and antitermination in E.coli

大肠杆菌中的转录终止和抗终止

• 批准号: 7095948
• 负责人: RANJAN SEN
• 金额: $ 5.27万
• 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/7095948
• 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，而负责转录的酶 RNA 聚合酶 (RNAP) 是不同微生物病原体中有吸引力的药物靶点。在给定时间，大多数 RNAP 分子参与 mRNA 合成和快速周转，这涉及转录中的两个关键步骤，即延伸和终止。因此，理想的药物靶标应该是参与这两种模式的 DNA 结合 RNAP 分子，而不是它们在细胞质中的游离形式。该项目的长期目标是了解转录过程的延伸、终止和反终止步骤的机制，以便未来合理的药物设计成为可能。主要重点是阐明 RNAP 的活性位点动力学以及这些步骤中复杂的蛋白质-DNA-RNA 相互作用。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物中蛋白质-DNA-RNA 相互作用以及了解终止/抗终止过程机制的理想系统。体内研究表明，携带志贺毒素的lamdoid噬菌体H19B需要噬菌体因子N和宿主因子NusA来修饰延伸复合物并实现抗终止。因此，这种抗终止复合物对于生化和结构研究来说要简单得多。这种修饰的伸长复合物中的相互作用将通过诱变、Fe-BABE 裂解和荧光光谱来表征。 RNA 聚合酶上 N 结合表面的 3D 定位将通过基于 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来获得。在平行研究中，将通过使用化学切割、足迹印迹、交联和荧光来实验研究 RNAP 响应不同 DNA 序列、新生 RNA 结构和反式因子（如 N 蛋白等）的活性位点动态。光谱学。分子动力学模拟等计算方法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伙伴。了解活性位点动力学将为未来合理的药物设计奠定基础。

项目成果

Escherichia coli RNA polymerase mutations located near the upstream edge of an RNA:DNA hybrid and the beginning of the RNA-exit channel are defective for transcription antitermination by the N protein from lambdoid phage H-19B.

大肠杆菌 RNA 聚合酶突变位于 RNA:DNA 杂交体的上游边缘和 RNA 退出通道的起点附近，对于来自 Lambdoid 噬菌体 H-19B 的 N 蛋白的转录抗终止有缺陷。

• 发表时间: 2005-09-09
• 作者: Cheeran, Anoop;Babu Suganthan, Rajan;Swapna, G;Bandey, Irfan;Achary, M Sridhar;Nagarajaram, H A;Sen, Ranjan
• 通讯作者: Sen, Ranjan

The site of action of the antiterminator protein N from the lambdoid phage H-19B.

来自 Lambdoid 噬菌体 H-19B 的抗终止子蛋白 N 的作用位点。

• 发表时间: 2007-10-19
• 作者: Cheeran, Anoop;Kolli, Nanci R;Sen, Ranjan
• 通讯作者: Sen, Ranjan