Gel-based fluorescence resonance energy transfer (gelFRET) analysis of nucleoprotein complex architecture

Abstract

A gel-based fluorescence resonance energy transfer (gelFRET) assay was developed for analysis of the architecture of nucleoprotein complexes. gelFRET is based on fluorescence analysis of nucleoprotein complexes separated by polyacrylamide gel electrophoresis. These complexes are separated from free components and nonspecific complexes, enabling fluorescence analysis of complexes containing all components in stoichiometric proportions. gelFRET can be used to investigate the structural organization of nucleoprotein complexes through comparison of the relative efficiencies of energy transfer from donor fluorophores linked to different positions on DNA to an acceptor fluorophore linked to a unique position on the binding protein. We have applied gelFRET to analysis of the orientation of binding by heterodimeric transcription factors. By using Fos-Jun heterodimers as a model system we have identified the structural determinants that control the orientation of heterodimer binding. gelFRET can be applied to studies of a variety of biological processes that influence the proximity of two sites within a complex, such as the assembly of transcription regulatory complexes.