Initiation of Transcription by T7 RNA Polymerase

这些研究详细描述了T7 RNA聚合酶野生型和修饰型启动子的启动子结合和转录起始机制。在启动子内使用核苷酸模拟报告的停流荧光研究，结合单次或有限周转猝灭流研究中RNA合成的直接测量，将提供定量动力学分析。初始目标将是阐明沿起始途径的速率限制步骤。使用有序加成动力学实验，起始中单个步骤的温度依赖性，以及在启动子中引入特定的结构扰动，将提供对导致第一个磷酸二酯键合成的单个步骤的深入了解。在这些研究的同时，将采取各种方法来表征开放配合物的结构和动力学。足迹将与光谱探针相结合，以表征开放复合物的程度和聚合酶与启动子的单个模板和非模板链之间相互作用的性质。这些相互作用的时间过程将与RNA合成的时间过程进行比较，以完成机制图。为了更完整地描绘转录起始的复杂机制，将利用温度、限制组分和模板扰动来改变速率限制步骤。新的速率决定步骤将与以前一样具有特征，以提供关于起始途径关键要素的机制细节。先前已经通过功能基诱变和稳态动力学分析制备了启动子的识别接触图谱。在初步确定起始途径的各个步骤之后，将评估一些先前确定的启动子接触对每个步骤的贡献。这将提供这种或任何其他RNA聚合酶的结构和机制之间的第一个详细的相关性。在转录起始位点附近，通过将简单的碱基类似物、碱基位点和替代连接物结合到启动子DNA中，将探索特定DNA功能基团在DNA模板在活性位点定位中的作用。随着替代越来越多的干扰，将分析起始选址的保真度。降低起始位点保真度的修饰也可能干扰起始的动力学步骤。这些将被动力学表征，以便将模板定位与特定的启动机制联系起来。将开发工具来跟踪底物核糖核苷酸在起始过程中与DNA模板的结合。这种方法将用于表征初始核糖核苷三磷酸的结合，但后来可能证明在转录的后续阶段监测底物结合是有用的。对复制细菌病毒基因组的酶T7 RNA聚合酶进行生物分子机制研究，涉及与聚合DNA序列相关的多亚基酶复合物的关联。这些研究将揭示参与病毒基因组复制过程早期阶段的分子相互作用。该酶识别的DNA序列的遗传和生化操作将通过复杂的方法进行研究，以产生与DNA上特定序列相关的复合物组分的详细分子快照系列，并产生聚合RNA序列。这些研究将有助于详细了解这种简单的酶复合物如何实现RNA合成过程，并将其应用于更复杂的RNA聚合酶的研究。＊＊＊

9630447 Martin These studies address a detailed characterization of the mechanisms of promoter binding and transcription initiation on wild type and modified promoters of T7 RNA polymerase. Stopped-flow fluorescence studies using nucleotide analog reporters within the promoter, combined with the direct measurement of RNA synthesis in single or limited turnover quench-flow studies will provide quantitative kinetic analyses. An initial goal will be the elucidation of the rate limiting step(s) along the initiation pathway. The use of ordered-addition kinetic experiments, the temperature dependence of individual steps in initiation, and the introduction of specific structural perturbations in the promoter will provide insight into the individual steps leading to the synthesis of the first phosphodiester bonds. In parallel with these studies, various approaches will be taken to characterize the structure and dynamics of the open complex. Footprinting will be combined with spectroscopic probes to characterize the extent of the open complex and the nature of the interactions between the polymerase and the individual template and nontemplate strands of the promoter. The time course of these interactions will be compared with the time course of RNA synthesis to complete the mechanistic picture. In order to more completely map the complex mechanism of transcription initiation, temperature, limiting components, and template perturbations will be exploited to alter the rate limiting step. The new rate determining step will be characterized as before, to provide mechanistic detail on key elements of the initiation pathway. A map of recognition contacts along the promoter has been previously prepared via functional group mutagenesis and steady state kinetic analyses. Following the preliminary identification of individual steps along the initiation pathway, the contributions of some of the previously identified promoter contacts to each of these steps will be assessed. This will provide the first de tailed correlations between structure and mechanism in this or any other RNA polymerase. Near the start site for transcription, the roles of specific DNA functional groups in the positioning of the DNA template at the active site will be explored by the incorporation into the promoter DNA of simple base analogs, abasic sites, and vicarious linkers. The fidelity of start site selection will be analyzed as substitutions become increasingly more perturbing. Modifications which decrease the start site fidelity may also be expected to perturb kinetic steps in initiation. These will be characterized kinetically in order to tie template positioning to specific mechanisms for initiation. Tools will be developed to follow the binding of substrate ribonucleotides to the DNA template during initiation. This approach will be used to characterize the binding of the initiating ribonucleoside triphosphates, but later may prove useful in monitoring substrate binding in subsequent stages of transcription. %%% Biomolecular mechanistic studies of the enzyme that replicates the genome of a bacterial virus, T7 RNA polymerase, are proposed involving the association of a multisubunit enzymatic complex associating with polymeric DNA sequences. These studies will reveal the molecular interactions involved in the early stages of the replication process of the viral genome. Genetic and biochemical manipulation of the DNA sequences recognized by the enzyme will be studied by sophisticated methods to yield a detailed series of molecular snapshots of the components of the complex associating with particular sequences on DNA and producing polymeric RNA sequences. These studies will contribute to a detailed understanding of how the process of RNA synthesis is achieved by this simple enzyme complex for application to the study of more complicated RNA polymerases. ***