Development and application of a quantitative analysis method for fluorescence resonance energy transfer localization experiments

This thesis focuses on Bayesian data analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments carried out to infer structural information of biological macromolecules and their complexes labeled with fluorescent dyes. In general, the measurement of FRET efficiencies allows to determine the distance of two dyes on the nanometer length scale. Moreover, it is possible to calculate the yet unknown position of one or more dyes relative to each other by trilateration. In doing so, FRET efficiency measurements between the dye to be localized, called antenna, and several dyes at known locations, called satellites, are used to constrain the position of the antenna. This, in turn, can be used to answer questions in structural biochemistry, when, for example, the antenna is attached to a yet unlocalized constituent of a macromolecular complex, while the satellites are linked to positions known from X-ray crystallography experiments. However, a simple conversion of FRET efficiencies into distances is inapplicable since FRET efficiency depends also on the unknown orientation of the transition dipole moments of the dyes. To account for these effects, the Nano-Positioning System (NPS) was developed as a new tool for structural biochemistry. NPS applies Bayesian data analysis to infer possible positions of dyes and optionally also the positions and orientations of the subunits of a macromolecular complex. NPS was successfully tested by localizing a dye attached to a known position in yeast RNA polymerase II (Pol II) elongation complexes (ECs). It was then applied to study the influence of the transcription factor IIB (TFIIB) on the position of the nascent RNA and to map the pathway of the nontemplate and upstream DNA in yeast Pol II ECs. Furthermore, the position and orientation of the TATA binding protein (TBP) in initial transcribing complexes of Pol II were inferred. A deeper understanding of NPS was obtained by analyzing synthetic data. Unknown dye orientations were found to be the major source of localization uncertainty under commonly encountered experimental conditions. Synergy effects emerging from the simultaneous analysis of a FRET network containing several antenna and satellite dyes were observed to improve the accuracy of the inference. It is proposed to use FRET anisotropy data in addition to the commonly measured FRET efficiencies to calculate accurate dye orientations and thus dramatically improve localization.