Congrats Courtney and the team! Very interesting work.

Thanks!

Thanks!
We don't know specifically about the sliding length - but that's definitely something we would like to look at.
And thank you for reminding me about your paper, very relevant and interesting.

n/
www.biorxiv.org/content/10.1...
technion.bsky.social

#Transcription #TranscriptionFactors #IDPs #SingleMolecule #Biophysics
#IDRs #IntrinsicallyDisordered #OpticalTweezers #FacilitatedDiffusion
#DNAunzipping

18/
Kudos to Nir Strugo (nirstrugo.bsky.social)‬ who led the work, with help from Carmit Burstein, Noam Nago, and Hadeel Khamis, and also Saddam Hossein from the Hoffman Lab.
Thanks to Hagen Hofmann, Naama Barkai, Moshe Goldsmith, Gabi Rosenblum and vmindel.bsky.social��� for comments and/or help!

17/
Bottom line:
Using single-molecule assays, we show that IDRs modulate Msn2 binding to its target motif by tuning genome exploration.
This occurs via non-specific (or rather quasi-specific) association and sequence-sensitive facilitated diffusion, shaped by disordered regions.

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.

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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.

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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.

10/
Since IDRs increased both non-specific binding and association to the motif, we asked:
Can these non-specifically bound proteins ultimately reach the motif by sliding on DNA?

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.

8/
Based on these assays, and complementary EMSA and mass photometry experiments, we could conclude that IDRs promote cooperative formation of non-specific multimeric Msn2–DNA complexes, which are further stabilized by the DBD.