Single-Molecule Insights on the Human RNA Polymerase II Transcription Regulation: Assembly of the Pre-Initiation Complex, Re-Initiation Scaffold, and the Roles of Activators

Abstract

Transcription of all protein-coding genes in human cells begins with the assembly of the RNA polymerase II pre-initiation complex (PIC) composed of more than forty polypeptides (total size 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 conventional biochemical studies. We have developed a surface-based, promoter-specific Pol II transcription system suitable for single-molecule work. The system consists of (a) an imaging surface and a fluidics system supporting Pol II transcription from immobilized DNA templates; (b) site-specific labeling of recombinant and complex transcription factors; and (c) novel probes capable of single-molecule, real-time detection of mRNA synthesis. We have simultaneously imaged thousands of single-molecule transcription events at sub-second time resolution for hour-long time periods with two separate multi-color fluorescence imaging techniques: (a) actively stabilized (drift of <1 nm over hours) temperature-controlled total internal reflection (TIR) microscopy; and (b) the zero mode waveguide multiplex confocal imaging system developed by Pacific Biosciences. With this experimental setup, we have observed up to eight rounds of promoter-specific Pol II transcription per DNA template. The efficiency of transcription re-initiation was found much higher than the efficiency of the first transcription round, suggesting a “scaffold” left from the first round could facilitate re-initiation. We have further found that a key component of this scaffold is the promoter recognizing TFIID complex. Using multiple fluorescently labeled GTFs (TFIIB TFIID, TFIIF, Pol II, and TFIIE) we are currently investigating the structure and regulation of this re-initiation scaffold, the order of the PIC assembly, and the mechanism of transcription modulation by sequence-specific activators in the context of promoter DNA elements.