16/
Together, our results support a model in which IDRs:
1. Facilitate initial non-specific association, stabilized by the DBD. Association, stabilization, or both, are sensitive to the sequence.
2. Enhance sequence-dependent diffusion toward the motif.

15/
What about the diffusion? sequence-sensitive ?
We perturbed IDR function during the sliding phase only (post-binding).
This had no effect for the arb. seq. but reduced STO probability and delayed detection for Hap4
⇒ IDRs enhance diffusion in a sequence-sensitive manner.

14/
Can we pinpoint which specific phase of the search is sequence-sensitive?
Hap4 showed increased non-specific binding, while dissociation rates (very low for both environments) were similar.
Conclusion: initial association, but not dissociation, is sequence-sensitive.

13/
Can this mechanism explain Msn2’s promoter selectivity?
We tested by replacing our "arbitrary" flanking region with a segment from the Hap4 promoter (a native Msn2 target).
Strikingly, STO binding increased to ~100%, and TFs were detected faster.

12/
Surprisingly, TFs were detected at the motif in ~30% of molecules, despite no free TFs in solution and irreversible dissociation conditions. This required intact IDRs, supporting a search mechanism based on non-specific binding and 1D diffusion on DNA.

11/
To test this, we developed a new assay, which we called Sliding-to-Target Occupation (STO):
We unzip DNA, incubate with TFs for 1 min, and then move to a TF-free channel where we perform repeated unzipping cycles to detect binding at the motif.

9/
We also found that Msn2 interacts with single-stranded DNA through its IDRs. This was evident in rezipping hysteresis, EMSA, and the kinetics of DNA hairpin closing.
These interactions may be relevant for binding melted promoter regions during activation.

7/
During unzipping, we also detected non-specific binding events, evident as peaks far from the canonical motif.
These were frequent with full-length Msn2, rare with the DBD alone, and absent with the IDRs only or in protein-free controls.

6/
So IDRs affect the affinity, but is this due to a change in association or dissociation rate?
With our previously developed fluctuation assay (Khamis 2021), we saw that IDR deletion didn't affect k_{off} but reduced k_{on} 6-fold
⇒IDRs enhance association, not stability.

5/
Charge-mediated interactions mediate IDRs contribution: Adding free L-arginine reduced binding, but the effect was reversed at pH 9.8, where arginine is neutral. Notably, the DBD-only variant was less affected, giving us a tool to selectively perturb IDRs.

4/
Removing the IDRs sharply reduced both the probability and strength of binding at the recognition motif.
This suggests that IDRs enhance binding affinity.

3/
Msn2 has a canonical zinc finger DNA-binding domain (DBD) flanked by long IDRs.
What role do these IDRs play in DNA binding?
We used a single-molecule DNA unzipping assay capable of detecting DNA-bound proteins, and three variants: WT, DBD-only, and IDR-only.

16/
Together, our results support a model in which IDRs:
1. Facilitate initial non-specific association, stabilized by the DBD. Association, stabilization, or both, are sensitive to the sequence.
2. Enhance sequence-dependent diffusion toward the motif.

15/
What about the diffusion? sequence-sensitive ?
We perturbed IDR function during the sliding phase only (post-binding).
This had no effect for the arb. seq. but reduced STO probability and delayed detection for Hap4
⇒ IDRs enhance diffusion in a sequence-sensitive manner.

14/
Can we pinpoint which specific phase of the search is sequence-sensitive?
Hap4 showed increased non-specific binding, while dissociation rates (very low for both environments) were similar.
Conclusion: initial association, but not dissociation, is sequence-sensitive.

13/
Can this mechanism explain Msn2’s promoter selectivity?
We tested by replacing our "arbitrary" flanking region with a segment from the Hap4 promoter (a native Msn2 target).
Strikingly, STO binding increased to ~100%, and TFs were detected faster.

12/
Surprisingly, TFs were detected at the motif in ~30% of molecules, despite no free TFs in solution and irreversible dissociation conditions. This required intact IDRs, supporting a search mechanism based on non-specific binding and 1D diffusion on DNA.

11/
To test this, we developed a new assay, which we called Sliding-to-Target Occupation (STO):
We unzip DNA, incubate with TFs for 1 min, and then move to a TF-free channel where we perform repeated unzipping cycles to detect binding at the motif.