Large-Scale Conformational Changes in Regulation of Transcription Initiation

描述(申请人提供)：DNA依赖的RNA聚合酶(RNAP)在启动子DNA序列上的转录启动涉及结合自由能驱动的大规模构象变化。对具有代表性的E，Coli酶的研究的长期目标是表征这些构象变化的序列，并确定它们在创造转录泡和RNA合成活性部位中的作用。在下一个项目期间，将结合低分辨率结构特征(化学和酶足迹；荧光共振能量转移)进行结合和解离的动力学研究，以确定每一步发生什么构象变化，以及每一步如何为下一步奠定基础。主要的具体目标包括阐明从起始点上游60-80个碱基对的非特异性DNA序列的包裹如何极大地加速开放复合体的形成，以及识别RNA聚合酶关键区域的重排何时发生(例如，sima70掩蔽域从活性位点通道中排出，聚合酶非结构化区域的折叠)。为此，将描述野生型和缺失聚合酶突变体在删除上游或下游序列的启动子变体上形成的开放复合体的特征。具有相似大规模构象变化的重要但更简单的系统将被并行研究：2个转录因子(乳胶抑制物二聚体头饰，IHF)的特异性结合；以及2个类核蛋白(HU，IHF)的非特异性结合。温度和溶液变量对缠绕的IHF-H‘DNA界面形成热力学的影响将通过等温滴定量热法(ITC)和FRET来测量，以获得和解释DNA缠绕的热力学特征。多种结合方式和协同性在HU非特异性结合中的作用将用ITC来确定。通过ITC、圆二色谱和带有完整的Lac阻遏物四聚体的竞争硝酸纤维素滤膜分析，将研究局部折叠和蛋白质界面的形成与操作者结合的偶联。转录启动机制的研究进展影响人类健康的两个主要领域。首先，了解转录网络如何对发育和环境线索做出反应，对于识别和解决导致癌症等人类疾病的监管失误至关重要。第二，由细菌感染(例如结核病)引起的人类疾病对公众健康造成严重破坏。这一提案产生的结果将定义细菌启动的特定方面，从而揭示下一代抗生素的新目标。

DESCRIPTION (provided by applicant): Transcription initiation by DNA-dependent RNA polymerases (RNAP) at promoter DNA sequences involves large-scale conformational changes driven by binding free energy. The long term goal of this research on the representative E, coli enzyme is to characterize the sequence of these conformational changes and to determine their roles in creating the transcription bubble and active site for RNA synthesis. In the next project period, kinetic studies of association and dissociation will be performed in concert with low resolution structural characterizations (chemical and enzymatic footprinting; fluorescence resonance energy transfer (FRET)) to determine what conformational changes occur in each step, and how each sets the stage for the next. Key specific aims include elucidating how wrapping of nonspecific DNA sequences 60-80 base pairs upstream from the start site greatly accelerates open complex formation, and identifying when rearrangements in key regions of RNA polymerase (e.g. ejection of sigma70 masking domain from the active site channel, folding of unstructured regions of polymerase) occur. To this end, open complex formation by wild type and deletion polymerase mutants at promoter variants that delete upstream or downstream sequences will be characterized. Significant but simpler systems exhibiting analogous large-scale conformational changes will be studied in parallel: specific binding of 2 transcription factors (lac repressor dimer headpiece, IHF); and nonspecific binding of 2 nucleoid proteins (HU, IHF). Effects of temperature and solution variables on the thermodynamics of formation of a wrapped IHF-H' DNA interface will be measured by isothermal titration calorimetry (ITC) and FRET to obtain and interpret the thermodynamic signatures of DNA wrapping. Roles of multiple binding modes and cooperativity in nonspecific binding of HU will be determined using ITC. Coupling of local folding and formation of protein interfaces to operator binding will be investigated for the crosslinked dimeric headpiece by ITC, circular dichroism and a competitive nitrocellulose filter assay with intact lac repressor tetramer. Advances in the understanding of the mechanism of transcription initiation impacts 2 main areas of human health. First, understanding how transcriptional networks respond to developmental and environmental cues is fundamental to identifying and addressing failures in regulation that cause human diseases such as cancer. Secondly, human illnesses caused by bacterial infection (e.g. tuberculosis) wreak havoc on public health. Results generated by this proposal will define bacteria-specific aspects of initiation, and thus, reveal novel targets for the next generation of antibiotics.