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.