DYNAMICS OF T7 RNA POLYMERASE TRANSCRIPTION

描述：作为细胞大分子机械的结构信息 积累，我们的注意力转向理解这一动态 机械设备。对于多步骤反应，例如转录，我们想知道 大分子复合体是如何从一个步骤移动到另一个步骤的 反应途径。当存在替代路径时(即，暂停或不暂停；终止 或继续；取代RNA或形成用于启动复制的杂交) 要了解是什么决定了在这些途径之间的选择，以及 已经做出了决定。最后，我们想了解这些反应机制是如何 可能对监管很敏感。尽管两者在结构上的相似性有限 多亚基和单亚基RNAPs，转录反应由 这些酶非常相似，甚至在细节上也是如此，例如 发生启动子释放的RNA或终止子的精细结构， 表明这些结构不同的分子已经汇聚到 执行转录反应的类似解决方案。我们建议使用 结构特征良好的单亚基17RNAP作为理解的模型 转录的构象动力学。我们将使用核酸酶和FeBABE 结合RNAP探测RNAP：RNA/DNA的变化 当聚合酶从起始移动到延伸时发生的相互作用。 停顿时延伸物构象的变化，NTP 结合或形成扩展的杂交物也将具有类似的特征。 将使用工程设计的二硫化物交联剂来测量 RNAP功能的构象限制。序号和序号的作用 超螺旋作用于阻止RNA是否被移位，或者是否 扩展的或持久的杂交形式，将被定义。特定于序列 将确定特定站点暂停所需的交互。这个 活性部位的构象状态，通过T7的结合来调节 当RNAP暂停时，将用羧基肽酶监测溶菌酶。这个 启动子解离和RNA置换的机制将由 突变RNAP元件被认为对这些过程很重要， 并通过绘制这些元素所产生的DNA/RNA相互作用图。最后， T7RNAP启动T7DNA复制的机制将被研究以揭示 参与延伸的RNAP如何将启动转录本转移到DNA 聚合酶。

DESCRIPTION: As structural information on the cell's macromolecular machinery accumulates, our attention turns towards understanding the dynamics of this machinery. For a multi-step reaction, such as transcription, we want to know how the macromolecular complex moves from one step to another along the reaction pathway. When alternate pathways exist (i.e., pause or not; terminate or continue; displace the RNA or form a hybrid for priming replication) we want to understand what determines the choice between these pathways and how the decision is made. Finally, we want to understand how these reaction mechanisms may be sensitive to regulation. Despite limited structural similarity between the multi-and single subunit RNAPs, the transcription reactions mediated by these enzymes are remarkably similar, even in details such as the length of the RNA at which promoter release occurs or the fine structure of terminators, suggesting that these structurally dissimilar molecules have converged upon similar solutions for executing a transcription reaction. We propose to use the structurally well characterized single-subunit 17RNAP as a model to understand the conformational dynamics of transcription. We will use nucleases and FeBABE conjugated RNAPs to probe the changes in RNAP:RNA/DNA interactions that occur as the polymerase moves from initiation to elongation. The changes in elongation complex conformation which accompany pausing, NTP binding, or formation of extended hybrids will be similarly characterized. Engineered disulphide cross-links will be used measure the effects of conformational restriction on RNAP function. The roles of sequence and supercoiling in deterrr aboutining whether the RNA is displaced, or whether extended or persistent hybrids form, will be defined. The sequence-specific interactions required for site-specific pausing will be identified. The conformational state of the active site, which is regulated via binding of T7 lysozyme, will be monitored with carboxypeptidase as the RNAP pauses. The mechanisms of promoter unwinding and RNA displacement will be characterized by mutagenizing the RNAP elements proposed to be important for these processes, and by mapping the DNA/RNA interactions made by these elements. Finally, the mechanism by which T7RNAP primes T7 DNA replication will be studied to reveal how an RNAP engaged in elongation transfers a priming transcript to a DNA polymerase.