DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability

摘要

Schematic overview of the DNA origami force clamp. The ssDNA spring protrudes from the DNA origami body and spans the 43 nm gap of the rigid DNA origami clamp body (grey). Centred within the ssDNA spring is a double-stranded promoter region including the TATA-box element (blue) flanked by a donor/acceptor (green/red) fluorescent dye pair for FRET sensing. The ssDNA spring length can be adjusted with DNA from the reservoir by using different staple strands (teal/orange) during assembly. Reducing the number of nucleotides spanning the gap leads to a smaller number of adoptable conformations of the ssDNA chain and thus results in a higher entropic force. The length of the ssDNA spring for low (AdMLP: 3.3 pN/U6 promoter: 2.6 pN) and high forces (AdMLP: 6.0 pN/U6 promoter: 6.6 pN) is shown. Single-pair FRET assay as readout for the bending of promoter DNA by the TATA-binding protein (TBP, yellow). A donor (ATTO 532, green) and acceptor fluorophore (ATTO 647 N, red) flank the TATA-box element (blue), resulting in a low efficiency FRET between both dyes. Binding of TBP bends the DNA by ~90°, thereby decreasing the distance between the fluorophores resulting in an increase in FRET efficiency (DNA/TBP structures adapted from PDB: 5FUR).