Mechanisms of RNA Polymerase-Promoter and lac Repressor-Operator Interactions

 描述(申请人提供)：这项研究的一个直接目标是确定E.ColiRNA70RNA聚合酶和启动子σ的一系列大的构象变化，这些变化将最初的特异(封闭)复合体转化为不稳定的开放复合体，并在一些启动子处随后稳定这一最初的开放复合体。这些构象变化包括启动子DNA的大规模弯曲、包裹和开放以及RNAP活动元件的铰链弯曲、耦合折叠和组装。了解这些构象变化及其在启动机制中的作用，对于了解启动子序列和转录因子对启动的调控以及设计新的抗生素是必要的。本实验室用于研究动力学和表征这些构象变化的技术包括使用快速猝灭混合对放射性标记DNA进行快速足迹和过滤器结合分析，使用菁染料标记DNA的停流荧光动力学方法(FRET，PIFE)，以及测定和解释溶质和盐对机械步骤的速率和平衡常数的影响。溶质和盐对速率常数的影响提供了有关形成过渡态的构象变化和相互作用的信息，这是其他方法所不能获得的机制信息的来源。比较了RNAPσ70区1和启动子截断变异体与野生型RNAP和全长启动子的差异。我们正在检验这样的假设，即开放复合体的形成速度是由启动子特异性的差异调节的，这些差异在封闭复合体的系综中具有足够的先进性，使下游的双链弯曲到活性部位的裂隙中。这项研究的第二个直接目标是获得关键生化溶质与显示蛋白质和核酸官能团的模型化合物相互作用的热力学信息，这是解释溶质对速率和平衡常数的影响所必需的。 并对过渡态和中间体进行表征。采用蒸汽压渗透压法和溶解度分析法对小分子溶质(包括尿素和其他酰胺、甘油和其他多元醇)、渗透物质(包括甘氨酸甜菜碱、脯氨酸和海藻糖)以及一系列Hofmeister盐(从GuHSCN到Na2SO4)与显示生物聚合物官能团的模型化合物的优先相互作用进行了定量。对这些数据的新分析正被用来量化这些溶质与核酸和蛋白质的官能团的相互作用，以及各个官能团之间的相互作用。作为使用溶质来确定机制的试验，溶质对形成紫胶抑制复合体以及开放和稳定RNA聚合酶(RNAP)-启动子起始复合体的动力学的影响正在被确定并从机制的角度进行解释。通过基团相互作用的测定，获得了水中疏水(C-C)效应、N-酰胺O氢键、阳离子-π、-CH-π和O-酰胺C(n-π*)相互作用的新的定量信息。

 DESCRIPTION (provided by applicant): An immediate goal of this research is to determine the series of large conformational changes in E. coli σ70 RNA polymerase (RNAP) and promoter DNA that convert the initial specific (closed) complex to an unstable open complex and at some promoters subsequently stabilize this initial open complex. These conformational changes include large-scale bending, wrapping and opening of promoter DNA and hinge-bending, coupled folding and assembly of mobile elements of RNAP. An understanding of these conformational changes and their role in the initiation mechanism is necessary to understand regulation of initiation by promoter sequence and transcription factors, and to design new antibiotics. Techniques used in this laboratory to study the kinetics and characterize these conformational changes include fast footprinting and filter binding assays with radiolabeled DNA using rapid quench mixing, stopped flow fluorescence kinetic methods (FRET, PIFE) with cyanine-dye labeled DNA, and determination and interpretation of solute and salt effects on rate and equilibrium constants of mechanistic steps. Solute and salt effects on rate constants provide information about conformational changes and interactions in forming transition states, a source of mechanistic information not available by other methods. RNAP σ70 region 1 variants and promoter truncation variants are compared with wild-type RNAP and full-length promoters. We are testing the hypotheses that the rate of open complex formation is regulated by promoter-specific differences in the fraction of the ensemble of closed complexes that are sufficiently advanced to open, with the downstream duplex bent into the active site cleft. A second immediate goal of this research is to obtain the thermodynamic information on the interactions of key biochemical solutes with model compounds displaying protein and nucleic acid functional groups that is needed to interpret solute effects on rate and equilibrium constants and characterize transition states and intermediates. Vapor pressure osmometry and solubility assays are used to quantify preferential interactions of small solutes including urea and other amides, glycerol and other polyols, osmolytes including glycine betaine, proline, and trehalose, and the series of Hofmeister salts (from GuHSCN to Na2SO4) with model compounds displaying the functional groups of biopolymers. Novel analyses of these data are being used to quantify interactions of these solutes with the functional groups of nucleic acids and proteins, and interactions between individual functional groups. As tests of the use of solutes to determine mechanisms, solute effects on the kinetics of forming a lac repression complex and of opening and stabilizing the RNA polymerase (RNAP)- promoter initiation complex are being determined and interpreted in terms of mechanism. From determinations of group-group interactions, new quantitative information is obtained about the hydrophobic (C-C) effect, amideN-amideO hydrogen bonding, and cation-π, -CH-π and amideO-amideC (n-π*) interactions in water.