Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching

Abstract

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Figure 1.

High-speed optical tweezers for the study of protein–DNA interactions. (A) A single DNA molecule is suspended between two beads trapped in double optical tweezers. The DNA sequence contains target operator sequences for LacI (two copies of O1, purple, one copy of O3, green). A third bead, stuck on the coverslip, is coated with LacI at single molecule concentration (LacI, blue). A constant force, F_tot = ΔF, is alternatively applied to the right and left trap, making the DNA dumbbell move back and forth at constant velocity. The figure shows ΔF applied to the right trap and the corresponding direction of the dumbbell movement (green arrow). Force is kept constant over time by a double force-clamp feedback system, which measures force using quadrant photodiodes (QPD) and rapidly corrects trap positions through AOD. (B) A custom-built multichannel flow chamber allows efficient assembly of the molecular construct and buffer exchange. (C) Typical ultrafast force-clamp recording during LacI–DNA interaction. Alternating ±3 pN force (black signal) is applied to the DNA dumbbell, which moves in a ±200 nm range. Blue signal is the position of the DNA dumbbell versus time. The figure shows different time intervals in which LacI is either detached from the DNA molecule (unbound), sliding on DNA (sliding), or strongly bound to DNA (local interaction).

Figure 2.

LacI–DNA interactions. (A) Left: typical recording of the dumbbell position during LacI–DNA interaction. Right: position histogram. The spacing between long local interactions corresponds to the position of the three operators. Triangular wave is not visible due to the time scale. (B) Left: dumbbell velocity obtained from the position record in (A). Right: velocity histogram. In the absence of LacI–DNA interactions, the dumbbell moves at constant velocity (unbound); sliding events slow down the dumbbell (sliding) and local interactions with operators and with non-specific DNA sequences halt the system, as represented by the sharp peak around zero velocity (local interactions). (C) Representative position records of three subsets highlighted in panel (A) showing brief local interactions (green, A), switch from unbound to sliding and a long local interaction with O1 (magenta, B) and between sliding and a long local interaction with O3 (cyan, C). In all three panels, only positive forces are displayed. Similar plots are obtained for negative forces (not shown). F_tot = 3 pN, dumbbell oscillation range is ±200 nm.

Figure 3.

Duration and position of LacI local interactions along DNA. (A) Typical cumulative frequency distribution of event duration (black line) and double exponential fit (red line). The two rates k₁ and k₂ obtained from the fit correspond to the detachment rate of long and short events, respectively (k₁ = 19 ± 4 s⁻¹, k₂ = 368 ± 3 s⁻¹ for the data shown in figure). (B) Duration of events versus DNA position. Long and short interactions can be distinguished along the vertical axis. (C) Position histogram of local interactions. Long (red) and short (black) events were separated based on their duration (see methods). F_tot = 3 pN.

Figure 4.

Effect of IPTG on the detachment rates of long (A) and short (B) local interactions. Both panels represent the average rate before (gray) and after (red) addition of IPTG (k₁ = 6.4 ± 1.8 s⁻¹, n = 14 without IPTG, k₁ = 167 ± 42 s⁻¹, n = 12 with IPTG; k₂ = 1149 ± 107 s⁻¹, n = 44 without IPTG, k₂ = 1643 ± 298 s⁻1, n = 9 with IPTG; error bars = std). Student’s t-test applied, **P < 0.01. F_tot = 3 pN.

Figure 5.

LacI sliding velocity and diffusion coefficient along DNA. Panels A, D and G show three representative recordings of the dumbbell position versus time displaying different sliding velocities (500 ms duration, F_tot = 3 pN, dumbbell oscillation range is ±200 nm). Panels B, E and H show the corresponding average dumbbell velocity versus DNA position during: sliding only (cyan); sliding and local interactions (magenta); in the absence of interactions (green). Panels C, F and I show the corresponding LacI diffusion coefficient as a function of DNA position (blue).

Figure 6.

LacI sliding velocity correlates with DNA sequence. (A) Average sliding velocity versus DNA position for two recordings (measurement #1 and #2) obtained on the same LacI–DNA segment at different times. (B) Correlation analysis of the two measurements shown in panel A. The red curve shows linear regression on the data (black circles): y = a + bx, with a = 26 ± 1 nm/ms, b = 0.71 ± 0.02. The Pearson’s r-value (0.82) was taken as correlation value.

Figure 7.

Effect of force on the kinetics of local interactions. (A) Detachment rate of short interactions (k₂) as a function of force. Cyan curve is the fitting curve with the Bell’s bond model , which gives k₂⁰ = 350 ± 90 s⁻¹ and d₂ = 1.5 ± 0.2 nm. (B) Detachment rate of long interactions with the operator (k₁). k₁ is well fitted by a two-step model (magenta, adjusted R² = 0.95), whereas the Bell’s bond model cannot properly fit data (cyan, adjusted R² = 0.3). Fit parameters of the two-step model are (see ‘Materials and Methods’ section and Supplementary Figure S2): , , , , , , , .

Figure 8.

A model for allosteric regulation and target-search mechanism of LacI. LacI can adopt two main conformations, LacI-R* (blue), which weakly interacts with DNA and LacI-R (red) with high affinity for the operator. In the LacI-R conformation, the protein also slides along nonspecific sequences. IPTG drives the equilibrium toward the LacI-R* conformation, inhibiting LacI repression by strongly reducing both sliding and long interactions with the operator. k₁ and k₂ respectively represent detachment rates of long and short local interactions. Arrow lengths are representative of the equilibrium constant between states.