Direct observation of operator bypassing during rotation-coupled protein diffusion on DNA

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA. It is commonly assumed that proteins, while sliding, faithfully track the DNA grooves and bind their target sequence upon first encounter. However, proteins cannot easily discriminate between target and non-target stretches of DNA. To recognize its binding sequence upon first encounter, a DNA-binding protein must closely investigate each section of DNA it passes, causing this process to be prohibitively slow.  Since these proteins have been observed to bind relatively quickly, it can be inferred that in actuality there is some accuracy-speed trade-off inherent to the search.


We combined resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA.  To directly test this accuracy-speed trade-off, we used single-molecule fluorescence resonance energy transfer (smFRET) to observe LacI skipping over its target sequence. To enhance our understanding of target bypassing, we explored the nature of how faithfully LacI tracks along the helical path of the DNA versus the idea that it may leave the helical path and ‘hop’.  To do this, we tracked sliding LacI molecules with a confocal laser and simultaneously measured their fluorescence correlation spectra (FCS) on the microsecond timescale. Our combined analysis of the tracking FCS and single-molecule FRET data reveals a speed-accuracy trade-off during sliding, where LacI hops to a neighbouring groove twice per turn. The data suggest that the weak nature of non-specific protein-DNA interactions underlies operator bypassing and at the same time facilitates rapid sliding. Nonetheless, the overall probability to bind a target sequence before leaving the DNA strand is close to unity, since 1D diffusion is inherently redundant and individual sequences are revisited many times.