Resolving the kinetics of transcription factor recognition of the target DNA site via advanced single-molecule FRET spectroscopy.

Abstract

Transcription factors function by specifically binding to target DNA sites to initiate gene expression. Such DNA recognition process usually involves an interplay between high-affinity binding to the cognate sequence and low-affinity binding to any other DNA. The latter enables scanning of the DNA via a 1D diffusive process, which combined with 3D diffusion-collision kinetics, facilitates target location. 1D diffusion has been observed using single-molecule fluorescence tracking on many DNA binding proteins (DPBs). Powerful experimental methods are also available to determine the sequence logo recognized by any given DBP. However, there are no approaches available that resolve the kinetics of locking into the target site, which is essential to elucidate the mechanisms of cognate DNA recognition. Here we introduce such an approach, by using single-molecule FRET spectroscopy and maximum likelihood analysis of photon arrival times. We apply it to determine the rates of lock-into and release-from the target site of the Engrailed homeodomain (EnHD). To resolve between non-specific and cognate DNA binding we exploit the recent discovery that EnHD changes conformation upon cognate binding, resulting in a large shift in transfer efficiency between a donor-acceptor pair placed at the protein ends. Our experiments on a 21-bp dsDNA containing the EnHD cognate sequence reveal that EnHD finds its target in few milliseconds. Remarkably, the on-rate at the C50 is mostly invariant over orders of magnitude differences in ionic strength and DNA concentration. This is because the changes in affinity caused by ionic strength are mostly recapitulated on the off-rate, which is the exact opposite to diffusion-control kinetics. We hence conclude that EnHD's lock-into target kinetics are controlled by its conformational dynamics, which makes the response time for controlling transcription only dependent on site occupancy levels.