Human TBP Bending of DNA Measured at the Single Molecule Level

Abstract

TATA binding protein (TBP) binds to DNA and bends it as a critical step of eukaryotic transcription. Human TBP induces an ∼100° bend in the DNA helix. To help elucidate the role of DNA architecture in transcription, we developed a single-molecule, optical-trapping assay to study the TBP-induced bending of DNA. We hypothesized that DNA bending would lead to an apparent shortening of the DNA. The predicted signal size is small (∼5 nm at 1 pN) and expected to decrease with increasing force. Moreover, the dynamics of TBP are slow, with a measured off-rate of ∼10−2 s−1 at physiological salt conditions. To detect these small, infrequent events, we developed an actively stabilized optical trapping instrument. We achieved high spatiotemporal resolution [0.56 nm (Δf = 0.01-1 Hz) over 200 s] at low force (1 pN) by developing a vertical optical trapping assay using short DNA tethers (92 nm) along with small beads (330 nm dia.). Detection of distinct TBP and TATA-box dependent signals required DNA molecules engineered to contain only one consensus TATA-box with no TATA-box like sequences in the flanking DNA. Significant nonspecific binding to the glass surfaces was minimized by covalently attaching polyethylene glycol (PEG) to the glass. With these experimental procedures, we directly observed individual bending and unbending events. The TBP-dependent DNA conformational changes were dynamic on the timescale of tens of seconds at a [TBP] ≈ 20 nM and a force of 1 pN. The change in DNA extension (∼5 nm) agreed well with theoretical predictions at 1 pN, but, unlike the simplest theory, showed little change in magnitude as the force was decreased. We expect that this assay will be broadly useful for studying DNA-protein interactions at high spatiotemporal resolution.