Position of the non-template DNA in the Polymerase II elongation complex revealed using single-molecule Fluorescence Resonance Energy Transfer

Abstract

Single Molecule Fluorescence Resonance Energy Transfer (FRET) was used as a quantitative tool to localize non-template DNA in the RNA Polymerase II (Pol II) elongation complex.We measured the FRET efficiencies between ‘antenna dye molecules’ (ADMs) attached to the non-template DNA and several ‘satellite dye molecules’ (SDMs) attached to known positions within the Pol II elongation complex using the recently developed Nano Positioning System (NPS) (1). NPS combines x-ray crystallographic information, single-molecule FRET data and bayesian parameter estimation, a probability based analysis method, to compute the three dimensional probability density for the position of ADMs. Within the bayesian framework, we were able to account for errors in the determined Forster radii, errors in the measured FRET efficiencies and uncertainties in the SDM positions (due to the attachment of the SDMs via flexible linkers) as well as for geometric constraints.We determined positions of the non-template DNA at +1, -2, -4, -7 (within the transcription bubble) and -12, -15, -18 of the up-stream DNA. Together with the high resolution x-ray structure and our earlier studies (2) determining the position of the nascent RNA this study completes the picture of the Pol II elongation complex.1. A. Muschielok, J. Andrecka, A. Jawhari, F. Bruckner, P. Cramer and J. Michaelis, A nano-positioning system for macromolecular structural analysis. Nature Methods, accepted.2. J. Andrecka, R. Lewis, F. Brueckner, E. Lehmann, P. Cramer and J. Michaelis, Tracking the position of mRNA in RNA polymerase II elongation complexes. PNAS 105, 135-140 (2008).