激活复合体组装与顺式元件在σ70启动子上的几何分布的相关性研究

As a major process of the initiation of prokaryotic gene transcription, the cis elements on the σ70 dependent promoters, modulate protein-protein interaction of RNA polymerase and the transcriptional regulators, protein-DNA interaction of the transcriptional regulators and the promoter region, to recruit RNA polymerase for the assembly of transcription complex. The geometry of cis elements on the promoter region determines the interaction modes of RNA polymerase and the transcriptional regulators, as well as of the transcriptional regulators and the promoter region, and modulates the transcriptional initiation mainly by altering the threshold of the free energy of the transcription activation complex formation. In this project, the impact of the geometry of cis elements on the assembly of the transcriptional complex and initiation will be investigated by two approaches: Firstly, we plan to design two cis elements located tandemly upstream of the core promoter to mimic the complex modes of transcription within a genome. We will investigate the accommodation of the transcription activation complex with the alteration of the geometry of promoters caused by multi cis elements interaction, compared to single cis element interaction. Secondly, we plan to manipulate the length and composition of the amino acid residues of the inter-domain linker of alpha subunit of RNA polymerase, to investigate its contribution to the function of RNA polymerase. The outcomes of the above project will provide better understanding of the reason for the existence of multi-mode of the transcriptional regulation in a genome, as well as the correlation between the polymorphism of amino acid sequence of alpha subunit and the function of RNA polymerase in bacteria and plastids.

作为原核基因转录起始的重要模式，σ70依赖型启动子上的顺式元件，通过介导RNA聚合酶与转录调控蛋白以及转录调控蛋白与DNA的相互作用，对RNA聚合酶进行招募，形成有效的转录复合体。顺式元件在启动子上的几何分布决定了它对RNA聚合酶与调控蛋白、调控蛋白与DNA的相互作用的调节模式，主要改变转录复合体形成的自由能阈值来影响起始转录。本项目将研究以下两个方面：一方面，设计两种顺式元件在启动子上游串联移动，来模拟体内复杂的转录模式，藉此探究转录起始复合物在与多调控元件相互作用相较于单调控元件相互作用时，对于启动子几何构象变化的接纳程度；另一方面，从RNA聚合酶α亚基结构域间链接区出发，改变其长度和氨基酸组成，来了解该区对RNA聚合酶功能行使的影响。以上研究结果将帮助人们更好地理解基因组中不同的转录调控模式的合理性，以及不同细菌和植物叶绿体的α亚基序列多态性与功能的相关性。

本项目采用了合成生物学方法，系统地进行了激活复合体组装与顺式元件在sigma70启动子上的几何分布的相关性研究，揭示了启动子上CRP总体调控蛋白靶位点的空间依赖性的决定因素。本项目的研究结果有助于了解基因组水平上CRP等总体调控蛋白在不同靶基因启动子上的调控多样性。与此同时，我们尝试把本项目中已积累的研究经验和成果扩展到更广泛的基因表达调控研究中，如在研究铜绿假单胞菌总体调控蛋白CpxR对耐药性相关的mexAB-oprM操纵子的表达调控作用，取得了重要的研究进展。