A Single Molecule FRET Study of Formation of RNA Polymerase II Elongation Complex on Passivated Surface

RNA polymerase II (pol II) is an essential enzyme that transcribes protein-coding genes. It is of rudimentary and medical importance to understand the mechanisms of pol II transcription. By passivating fused-silica surface with biologically-friendly polyethylene glycol (PEG), we immobilized a RNA-primed double-stranded DNA, termed DNA/RNA scaffold, to build a single molecule transcription assay using FRET (fluorescence resonance energy transfer) signals from a donor on DNA and an acceptor on RNA as readouts, which allowed us to monitor how pol II assembles with the DNA/RNA scaffold with a total internal reflection fluorescence microscope. Here, our single molecular FRET assay shows that the DNA/RNA scaffold assumes heterogeneous conformations in the absence of pol II but retains a unique conformation in the presence of pol II, indicated by a single-valued FRET value consistent with the RNA engaging in the pol II's RNA exit channel. Our study of pol II interacting with DNA/RNA offers an example for demonstrating that the single FRET assay is powerful in the following aspects: (1) it improves the spatial resolution of a few hundreds of nanometers, limited by optical diffraction on an ordinary fluorescence microscope, to nanometers; (2) it provides dynamical information with temporal resolution set by fluorescence microscopy instrumentation; and (3) it allows sorting of single molecules to reveal the heterogeneity, averaged out by bulk measurements. Such a single molecule FRET transcription assay can be readily extended to visualize nascent RNA extension in real-time with the supply of NTPs, or be applied to dissect the architecture of various kinds of RNA polymerases, including pol I and pol III, the relatives of pol II.