SmFRET experiments of the RNA polymerase II transcription initiation complex.

Abstract

Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation. We present detailed protocols for the purification of all essential protein components of the minimal eukaryotic transcription initiation complex. Moreover, we demonstrate how elaborate strategies for site-specific protein labeling can be used to produce complexes with dye molecules attached to arbitrary desired positions. These complexes are then used for smFRET measurements. Moreover, we describe the Nano-Positioning System (NPS) which allows us to quantitatively use the results from a network of smFRET measurements to obtain structural information. With this we provide a toolbox to answer open questions which could not be addressed using methods like X-ray crystallography or cryo-electron microscopy.