RUI: Discovering and characterizing the binding determinants for RNA polymerase-associated proteins in E. coli

为了使细胞能够生长并对不断变化的环境条件做出谨慎的反应，细菌和所有细胞一样，需要确保它们的每个基因在适当的时间和适当的水平上表达。基因表达的第一步称为转录，由称为RNA聚合酶（RNAP）的分子机器介导，并由统称为转录因子的大量蛋白质指导。虽然对细菌RNAP的研究继续为理解生命所有领域的转录提供了一个框架，但缺乏对RNAP和转录因子相互作用的深入理解。在这项研究中，本科生科学家将使用经典和创新的分子生物学技术来发现和表征转录因子如何影响细菌E.杆菌这些发现将使人们更好地理解所有细胞中基因表达的基本原理，开发的实验工具将促进未来蛋白质-蛋白质相互作用的研究。学生科学家将在研究过程的各个方面得到指导。此外，研究人员将开发一个基于转录因子-RNAP相互作用的CURE（课堂本科生研究经验），在资助期间教授50名本科生，之后还有更多关于蛋白质-蛋白质相互作用的基本主题。该项目还将开发一个研究模块，纳入一个完善的科学教育推广计划，为高中女生，这将有助于他们为未来的STEM职业道路和科学领导角色做好准备。在E.在大肠杆菌中，它们如何影响基因表达，以及它们是否与RNAP或其七西格玛因子进行直接的蛋白质-蛋白质接触，仍有待发现。在这个项目中，研究人员将发现和表征转录因子-RNAP结构域相互作用和转录因子-sigma结构域相互作用。在RNAP晶体结构可用性的指导下，研究人员将来自RNAP的47个结构域及其sigma因子输入到细菌双杂交（B2 H）测定中。超过100个先前确定的候选RNAP相关蛋白将单独测试对每个RNAP和sigma因子域的相互作用。此外，RNAP和σ因子结构域将针对E.大肠杆菌蛋白质组文库，使用B2 H测定菌株，该菌株针对荧光激活细胞分选方案进行了优化，从而能够分离含有生产性蛋白质-蛋白质相互作用的细胞。研究人员将使用下一代测序来识别与相互作用的蛋白质-蛋白质对相对应的DNA。在创建RNAP和sigma因子相关蛋白的景观图之后，他们将通过鉴定转录因子和RNAP结构域中破坏相互作用的氨基酸取代来表征示例性相互作用。这些遗传工具将被用来了解更多关于RNAP功能的影响，通过调查的转录因子RNAP结构域的相互作用对已知的表型的转录因子在体内，以及在启动，延伸和终止性能的RNAP在体外转录。

To enable both cellular growth and prudent responses to changing environmental conditions, bacteria, like all cells, need to ensure that each of their genes is expressed at the appropriate time and at the appropriate level. The first step of gene expression, called transcription, is mediated by a molecular machine called RNA polymerase (RNAP) and is guided by a plethora of proteins collectively named transcription factors. While the study of the bacterial RNAPs continues to provide a framework for understanding transcription in all domains of life, an in depth understanding of RNAP and transcription factor interactions is lacking. In this research, undergraduate student-scientists will use classical and innovative molecular biology techniques to discover and characterize how transcription factors influence RNAP activity in the bacterium E. coli. The findings will lead to a greater understanding of the fundamental principles governing gene expression in all cells and the experimental tools developed will facilitate future studies of protein-protein interactions. Student-scientists will be mentored in all aspects of the research process. In addition, the investigators will develop a CURE (Classroom Undergraduate Research Experience) based on transcription factor-RNAP interactions to teach fifty undergraduates during the funding period, and many more afterwards, about the fundamental topic of protein-protein interactions. The project will also develop a research module for inclusion in a well-established science education outreach program for high school girls that will contribute to their preparedness for future STEM career paths and leadership roles in the sciences. For the great majority of the 250 transcription factors in E. coli, it remains to be discovered how they influence gene expression and if they make a direct protein-protein contact with RNAP or its seven sigma factors. In this project, the investigators will discover and characterize transcription factor-RNAP domain interactions and transcription factor-sigma domain interactions. Guided by the availability of RNAP crystal structures the investigators have inputted 47 structured domains from RNAP and its sigma factors into a bacterial two-hybrid (B2H) assay. Over 100 previously-identified candidate RNAP-associated proteins will be tested individually for interaction against each RNAP and sigma factor domain. Further, the RNAP and sigma factor domains will be screened against an E. coli proteome library using a B2H assay strain that is optimized for a fluorescence-activated cell sorting protocol enabling the isolation of cells containing productive protein-protein interactions. The investigators will use next generation sequencing to identify the DNA corresponding to the interacting protein-protein pairs. Following the creation of a landscape map of RNAP- and sigma factor-associated proteins, they will characterize exemplary interactions by identifying amino acid substitution(s) in both the transcription factor and the RNAP domain that disrupt the interaction. These genetic tools will be used to learn more about RNAP function by investigating the effect of the transcription factor-RNAP domain interaction on known phenotypes for the transcription factor in vivo, as well as on the initiation, elongation and termination properties of RNAP during transcription in vitro.