DNA binding proteins explore multiple local configurations during docking via rapid rebinding

Abstract

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.

Figure 1.

Single-molecule FRET assay for probing RT dynamics. (A) Two possible models describing flipping transition left- tumbling model, a basic three state model in which RT can undergo a transition of flipping or dissociation from a bound state and right- hopping model, a model with a pseudo bound state in which RT must undergo to the transition of flipping or dissociation through the pseudo state. (B) Schematic diagram of detection of two binding orientations of RT on a double primer DNA substrate. Larger green star and smaller red star represent a low FRET binding mode, larger red star and smaller green star represent a high FRET binding mode of freely diffusing Cy3(green sphere)-labeled RT to surface immobilized Cy5(red sphere)-labelled DNA. (C) FRET analysis of RT binding to a double-primer DNA substrate. Top: fluorescence time traces of Cy3 (green) and Cy5 (red) under 532 nm laser excitation. Bottom: FRET value calculated over the duration of the binding events. Arrows represent low-to-high (pink) and high-to-low(blue) FRET flipping events. Yellow shaded region identifies the bound state and (D) FRET distribution histogram of RT binding on 19-bp double primer DNA at 50 mM NaCl. The histogram was constructed from 1534 binding events.

Figure 2.

Binding kinetics of RT on DNA as a function of salt concentration. (A) Example FRET traces of RT binding dynamics on dpdsDNA. Top: binding dynamics at 50 mM NaCl, bottom: at 200 mM NaCl. Green and red represent a low FRET and high FRET bound states. Black line represents a flipping transition between the two bound states. Yellow shaded region identifies the bound state. (B) NaCl linkages ( versus ) for the binding of RT to 19-bp dpdsDNA. The slope of the linear fit (red line) to the data (squares) is 2.55, the thermodynamic net average number of ions released upon RT–DNA complex formation. (C) Salt concentration dependent binding rate calculated from the frequency of RT binding to DNA over the observation time. (D) Salt concentration dependent dissociation rate derived from the dwell times of RT binding. Dashed red line is a reference line connecting the data points. Number of binding events analyzed for 50, 100, 150 and 200 mM salt concentrations are 1534, 2700, 4431 and 659, respectively.

Figure 3.

Effects of increased macromolecular crowding on the binding kinetics of RT on dpdsDNA. (A) representative FRET traces of RT binding dynamics on dpdsDNA. Top: data obtained at 150 mM NaCl, bottom: data obtained at 150 mM NaCl and 10% (w/v) PEG 8K. Flipping (black line) of RT between low FRET (green) and high FRET (red) is pronounced under increased crowding. Yellow shaded region identifies the bound state. (B) Dissociation constant (K_d) of RT binding on DNA under macromolecular crowding. Red curve is a fit to the data (squares) based on scaled particle theory. Blue region is where the concentration of cellular crowding falls in which RT is estimated to have sub-nanomolar binding affinity. (C and D) Rate of binding and dissociation of RT binding on DNA as a function of PEG 8K at 150 mM NaCl, respectively. Dashed red line is a reference line connecting the data points. Number of binding events analyzed for 0, 2.5, 5, 7.5 and 10% (w/.v) PEG 8K concentrations are 4431, 3925, 4120, 911 and 1049, respectively.

Figure 4.

Binding dynamics of RT on 21-bp dpdsDNA with an incoming dNTP. (A) A 21-bp double primer DNA construct with a chain-terminating (ddC) priming end (double arrow) to study the effects of an incoming nucleotide on RT binding kinetics. (B) Representative FRET traces of RT binding dynamics on 21-bp double primer with a chain terminating dNTP. Top: RT binding under 150 mM NaCl, middle: 150 mM NaCl and 250 μM dTTP and bottom: 150 mM NaCl, 250 μM dTTP and 7.5% PEG 8K, respectively. Yellow shaded region identifies the bound state. (C) Dissociation constant of RT binding on 21-bp DNA-ddC. (D and E) Rate of binding and rate of dissociation of RT on 21-bp DNA-ddC. Number of binding events analyzed for RT, RT+dTTP and RT+dTTP+PEG 8K are 393, 284 and 471, respectively.

Figure 5.

Effects of increased salt concentration and macromolecular crowding on the flipping transition of RT. (A) NaCl dependence of probability of flipping of RT on 19-bp double primer. (B) NaCl dependence of rate of flipping. (C) PEG 8K dependence of probability of flipping of RT at 150 mM NaCl. (D) PEG 8K dependence of rate of flipping of RT at 150 mM NaCl. (E) Probability of flipping of RT on 21-bp double primer DNA-ddC (Figure 4A). (F) Rate of flipping of RT on 21-bp double primer.

Figure 6.

Free energy diagrams of RT flipping kinetics on DNA. The free energy diagrams were drawn for tumbling model and hopping model based on the kinetic and thermodynamic data obtained under different NaCl concentrations. (A) Free energy diagram corresponding to the tumbling model of RT binding and flipping kinetics under high salt concentration (black). Decreased salt concentration leads to increase in the free energy barrier height for dissociation transition and decrease in the barrier height for flipping transition relative to the bound or flipped bound state (orange). (B) Free energy diagram explaining flipping kinetics based on hopping model with an extra pseudo-bound state. In this free energy diagram both the bound state and flipped state are identical. Color convention is the same as in the free energy diagram for tumbling model.