Collaborative Research: Elucidating Pre-initiation Complex Assembly and Transcription Initiation by Pol-II Using Advanced Single Molecule and Microfluidic Methods

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

Intellectual Merit: RNA polymerase II (also called pol II) is an essential enzyme that catalyzes the transcription of DNA into messenger RNA (and most other forms of RNA), an important first step in gene expression. This project will elucidate key structural and functional aspects of the assembly of the Pre-Initiation Complex (PIC) of pol II and the mechanism of transcription initiation, i.e. the mechanistic steps involved in starting the transcription of a new gene. Sophisticated solution-based single-molecule fluorescence techniques, combined with microfluidics, will be applied to individual complexes in order to study how pol II assembles at a promoter (gene start site) and how it performs its catalytic function. Because single-molecule approaches remove ensemble and temporal averaging, information about the order and the kinetics of assembly will be unraveled. Broader Impacts: The "nano-biotechnology revolution" and the emergence of single molecule biophysics and super-resolution imaging have opened up tremendous opportunities in the investigation of processes that define life. One such process, fundamental to the "Central Dogma of Biology," is transcription. The approach for studying this basic process to be taken in this research could break conceptual and practical barriers that have hindered progress, and offers potential to catalyze discovery in many new ways. The tools and methods to be developed, as well as the general approaches pursued, will have greatest impact if distributed to a broad audience of receptive and innovative young scientists. Through educational and outreach programs, the project will expose a young audience (high school and college students), as well as more mature scientists, to novel research tools and methodologies that could be used to study many other biological processes and molecular machines.