Elucidating the Molecular Binding Mechanism of the Tata-Binding Protein using Pie-Pife

Abstract

The TATA-box binding protein (TBP) plays an important regulatory role in regulating gene expression by mediating the formation of the pre-initiation complex on promotor DNA. When binding to DNA, TBP melts the double helix and forms a stable complex while bending the dsDNA by ∼90o along the TATA sequence. In vitro single molecule experiments based on single-pair Förster resonance energy transfer (FRET) have recently revealed a dynamic, step-wise binding process via different intermediate conformations of the dsDNA. In vivo, TBP exists in different forms, e.g. as dimerbound to the dsDNA, or as part of different co-activator/deactivator complexes in the subsequent steps of pre-initiation complex formation. These states however are only observed at cellular concentration of TBP in the micromolar range, which is too high for in vitro single molecule fluorescence imaging. In this contribution, we present a new strategy to visualizes unlabelled TBP binding on DNA with changes in DNA conformation at high concentrations: binding of TBP is monitored via protein-induced fluorescence enhancement (PIFE) while single-pair FRET reports on ongoing conformational changes in the DNA/TBP complex simultaneously. We combine this label-free PIFE-FRET approach with pulsed interleaved excitation (PIE) spectroscopy, to investigate conformational dynamics of freely diffusing protein-DNA complexes in solution at the focus of a confocal microscope. Without the need of surface immobilization it can overcome concentration limitations of multi-colour assays by monitoring two parameters, binding and conformation rearrangement at the same.