arielkaplan.bsky.social
Single molecule biophysics, Optical Tweezers, Chromatin, Transcription, Polymerases, Helicases
Associate Professor, Technion-Israel Institute of Technology
https://kaplan.net.technion.ac.il/
40 posts
963 followers
218 following
Regular Contributor
Active Commenter
comment in response to
post
Thanks!
comment in response to
post
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.
comment in response to
post
n/
www.biorxiv.org/content/10.1...
technion.bsky.social
#Transcription #TranscriptionFactors #IDPs #SingleMolecule #Biophysics
#IDRs #IntrinsicallyDisordered #OpticalTweezers #FacilitatedDiffusion
#DNAunzipping
comment in response to
post
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!
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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?
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
4/
Removing the IDRs sharply reduced both the probability and strength of binding at the recognition motif.
This suggests that IDRs enhance binding affinity.
comment in response to
post
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.
comment in response to
post
2/
This work was inspired by studies from Naama Barkai’s lab at weizmanninstitute.bsky.social, showing that IDRs are essential for promoter-specific TF binding in vivo.
But what is the mechanism at the molecular level?
Do they contact DNA directly? How do they help with target search?
comment in response to
post
17/
Bottom line:
Using single-molecule approaches, we show that IDRs modulate Msn2’s binding to its target motif by tuning DNA exploration. This provides a potential mechanistic basis for promoter selectivity, perhaps general to IDRs-containing TFs.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
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.
comment in response to
post
Great work, congrats Beat and the team!
comment in response to
post
Interesting work. Congrats!
comment in response to
post
Congrats Tim!
comment in response to
post
Thanks!