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. 2012 Sep 18;109(38):15121-6.
doi: 10.1073/pnas.1208508109. Epub 2012 Sep 4.

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

Chandra P Joshi  1 , Debashis Panda, Danya J Martell, Nesha May Andoy, Tai-Yen Chen, Ahmed Gaballa, John D Helmann, Peng Chen
Affiliations

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

Chandra P Joshi et al. Proc Natl Acad Sci U S A. .

Abstract

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Dynamic CueR–DNA interactions. (A) Single-molecule EFRET trajectory of an immobilized Cy3-DNA interacting with holo-CueRCy5-C129 (2 nM). The cartoons on the right show CueRCy5-C129 in two binding orientations. The FRET donor (green sphere) and acceptor (red sphere) are drawn on the DNA and protein at their approximate locations. The gray line is original data; the red line is after nonlinear filtering. (B) Same as A, but with 2 nM holo-CueRCy5-E96C. (C) Same as A, but using the nonspecific DNA and 4 nM holo-CueRCy5-C129. Here, τ0 is denoted as τoff, and τ1 and τ2 are denoted together as τon. (D) Same as A, but with a mixture of holo-CueRCy5-C129 and holo-CueRCy5-E96C of 5 nM each. (E) Same as A, but with a mixture of apo-CueRCy5-C129 and holo-CueRCy5-E96C of 5 nM each. The blue arrows denote the transitions from the holo-protein bound states to the apo-protein bound states, and the black arrows denote the reverse transitions. (F) Histogram of EFRET trajectories of holo-CueRCy5-C129–DNA interactions at [holo-CueRCy5-C129] = 2 nM. Compiled from > 500 EFRET trajectories. Solid lines are fits of Voigt functions centered at about 0.07, 0.25, and 0.92, with percentage peak areas of 90.5 ± 0.1%, 4.9 ± 0.3%, and 4.6 ± 0.3%, respectively. Bin size, 0.01. (G) Same as F, but with 2 nM holo-CueRCy5-E69C. The three peaks center at about 0.07, 0.42, and 0.62, with percentage peak areas of 89.5 ± 0.1%, 5.5 ± 0.3%, and 5.0 ± 0.3%, respectively. (H) [CueRCy5-C129] dependence of the (E2 + E1)/E0 peak area ratios obtained from data such as in F. The solid lines are fits with Eqs. 8 (holo) and 9 (apo) (see also SI Appendix, section S15).
Fig. 2.
Fig. 2.
Single-step protein binding kinetics. (A) [CueR] dependence of formula image for CueRCy5-C129–DNA interactions. Solid lines are fits with Eq. 5 (see also SI Appendix, section S12). (B) Distributions of τ0 for holo- and apo-CueRCy5-C129 interactions with DNA, both at 0.5 nM protein concentration. Data compiled from about 500 (holo) and 600 (apo) EFRET trajectories. Bin size, 2 s. Both distributions were normalized to the first point for comparison. Solid lines are fits with a single exponential function Nγ exp(-γτ); γ(holo) = 0.038 ± 0.002 s-1, γ(apo) = 0.031 ± 0.004 s-1, and N is a scaling parameter.
Fig. 3.
Fig. 3.
Two different binding modes of CueR on DNA. (A) Distributions of τ2 for holo- and apo-CueRCy5-C129 interactions with specific DNA, both at 2 nM protein concentration. Data compiled from about 500 (holo) and 700 (apo) EFRET trajectories. Bin size, 0.15 s. The two distributions were normalized to the first data point for comparison. Solid lines are fits with a sum of two exponentials: N[Aγ1 exp(-γ1τ) + (1 - A)γ2 exp(-γ2τ)]. For holo: γ1 = 0.55 ± 0.01 s-1, γ2 = 6.4 ± 0.2 s-1, and A = 0.68 ± 0.01; for apo: γ1 = 0.35 ± 0.02 s-1, γ2 = 1.6 ± 0.06 s-1, and A = 0.57 ± 0.02. (B) Distributions of τ2→0 and τ2→1 at [holo-CueRCy5-C129] = 2 nM. Solid lines are global fits with a sum of two exponentials using the same parameters for the holo data in A. Bin size, 0.15 s. (C) Distribution of τon for holo-CueRCy5-C129 interactions with the nonspecific DNA at [holo-CueRCy5-C129] = 7 nM. To increase statistics, τ1 and τ2 from about 225 EFRET trajectories were combined here as τon. Bin size, 0.05 s. Solid line is a fit with Nγ exp(-γτ); γ = 12.8 ± 0.6 s-1.
Fig. 4.
Fig. 4.
Direct protein substitution and assisted dissociation. (A) [CueR] dependence of formula image for CueRCy5-C129–DNA interactions. Solid lines are fits with Eqs. 6 (holo) and 7 (apo). (B) The [holo-CueR] dependence of formula image for holo-CueRCy5-C129 interactions with the nonspecific DNA; the τ1 and τ2 are combined here and denoted as τon. Solid line is a fit with a horizontal line at 5.9 ± 0.1 s-1. Each data point in A and B is an average of data from about 250 EFRET trajectories. (C) Dependence of N2→1/N2→0 on [holo-CueRCy5-C129]. Solid line is a fit with Eq. 3. (D) Dependence of N2→1/N2→0 on [apo-CueRCy5-C129]. Solid line is a fit with Eq. 4. (Inset) Same data but the y-value is the inverse, N2→0/N2→1.
Fig. 5.
Fig. 5.
Mechanistic model of CueR–DNA interactions. Here, k’s are the kinetic constants and described in the text. P denotes protein.
Fig. 6.
Fig. 6.
Pathways for turning off transcription by CueR. The timescales are denoted for relevant kinetic steps, including (i) unbinding, (ii) binding, (iii) direct substitution, and (iv) assisted dissociation.
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