Structure and Relations of Proteins and Nucleic Acids

在这份续订申请中，我们描述了继续并完成几项正在进行的研究的计划 大肠杆菌转录复合体和噬菌体T4复制复合体的作用机制及调控。两者都有 代表了等同的基因表达“大分子机器”的最简单版本，它使用 在高等生物体中控制这些过程的大多数相同的成分。我们将特别关注 在模板导向的合成中，中心聚合酶如何执行单核苷酸加成循环，以及 这一过程如何被控制和重定向到另一种不太可能发生的动态竞争反应 在暂停站点、在终止站点以及作为以下结果的路径(包括终止和编辑) 残留物混入。我们还将重点介绍如何进一步调节这些主要的反应途径 通过调节因素，如抗终止复合体。为了支持这些总体目标，我们的具体目标 下一个报告所述期间的目标将是进一步审查以下问题：(1)制定和 开发一种新的光谱方法来研究局部RNA和DNA的构象和动力学； 进一步研究转录和复制中单核苷酸加成循环的步骤； 继续研究转录中反应途径选择的控制；(Iv)进一步了解 反终止系统的基本控制机制；(V)完成我们对RNA和蛋白质的模型研究 链环和动力学；(Vi)研究大肠杆菌转录相互作用的机制细节 终止因子Rho与RNA聚合酶；(Vii)继续我们对解旋酶机制和 转录和复制中的偶联；()进一步研究过程性钳制的机制 在噬菌体T4系统中装载；以及(Ix)继续我们对蛋白质-核酸相互作用的理论研究 运动学。这些研究应该有助于我们更好地理解这些复合体是如何组装的，以及 各组件相互作用，构建稳定且易于调节的“大分子机器”。就他们的 对于生物医学研究的意义，这些研究将继续作为功能和 控制高等生物的类似转录和复制系统，并可能有助于揭示 在癌症和其他基因表达不当的疾病中，这些复合体可能会出错。

In this renewal application we describe plans to continue and complete several ongoing studies on the mechanisms and control of the transcription complex of E. coli and the replication complex of phage T4. Both represent the simplest versions of the equivalent 'macromolecular machines' of gene expression that use most of the same components to control these processes in higher organisms. We will focus, in particular, on how the central polymerases perform the single nucleotide addition cycle in template-directed synthesis, and how this process is controlled and redirected into alternative and less probable kinetically competing reaction pathways (including termination and editing) at pause sites, at termination sites, and as a consequence of residue misincorporation. We will also focus on how these 'primary' reaction pathways are further modulated by regulatory factors, such as antitermination complexes. In support of these overall objectives, our Specific Aims during the next reporting period will be to further examine the following issues: (i) to develop and exploit a new spectroscopic method for studying local RNA and DMAconformations and dynamics; (ii) to further examine the steps in the single nucleotide addition cycle in transcription and replication; (iii) to continue to study the control of reaction pathway selection in transcription; (iv) to further understand the basic control mechanisms of antitermination systems; (v) to complete our model studies of RNA and protein chain looping and dynamics; (vi) to examine the mechanistic details of the interactions of E. coli transcription termination factor Rho with RNA polymerase; (vii) to continue our studies of helicase mechanisms and coupling in transcription and replication; (viii) to further examine the mechanisms of processivity clamp loading in the phage T4 system; and (ix) to continue our theoretical studies of protein-nucleic acid interaction kinetics. These studies should help us to better understand how these complexes assemble, and how the components interact to build stable, and yet easily regulatable 'macromolecular machines'. In terms of their significance for biomedical research, these studies will continue to serve as models for the function and control of the analogous transcription and replication systems of higher organisms, and may help reveal how these complexes can go awry in cancer and other diseases of inappropriate gene expression.