A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Automated summary

Transcription factors regulate gene activity by binding specific regions of genomic DNA through a complex interplay of specific and nonspecific interactions. The study utilized a label-free biosensor, Reflective Phantom Interface (RPI), to investigate Gal4's binding to DNA fragments and found that binding to its cognate site is stronger but slower than nonspecific binding. Analysis of binding curves and molecular dynamics simulations revealed a small free energy gap between specific and nonspecific binding, with a compensation of enthalpy and entropy in Gal4-DNA interactions.