Assembly and Regulation of the Human RNA Polymerase II Preinitiation Complex: A Single-Molecule Approach to a Complex Biological System

Abstract

Transcription of all protein-coding genes in human cells begins with the assembly of the RNA polymerase II preinitiation complex (PIC). The PIC consists of Pol II itself and at least six general transcription factors (more than forty polypeptides, total molecular weight of ∼ 3MDa). Due to the high complexity and the dynamic nature of the PIC, the mechanism of its assembly and regulation remains elusive after decades of biochemical studies. We have developed a surface-based, promoter-specific Pol II transcription system suitable for single-molecule analysis. The system consists of (a) an imaging surface and a fluidics system supporting Pol II transcription from immobilized DNA templates; (b) fluorescently labeled transcription factors (recombinant and highly purified from nuclear extracts); (c) novel probes capable of rapid real-time detection of mRNA synthesis at the single-molecule level. We have begun to use this system to dissect the PIC assembly process coupled to a fully reconstituted single molecule transcription reaction. We use two separate multi-color fluorescence imaging instruments capable of simultaneous detection of thousands of individual PIC assembly and transcription events at sub-second time resolution for hour-long time periods: (a) actively stabilized (drift of l1 nm over hours) temperature-controlled total internal reflection (TIR) microscope; (b) zero mode waveguide multiplex confocal imaging system developed by Pacific Biosciences. Using these first generation reconstituted single molecule RNA Pol II transcription systems, we have gained new insights into the mechanisms of human transcriptional regulation not attainable by the conventional in vitro biochemical assays that have been the gold standard for over 30 years.