Finally, CpG methylation shortened Egr-1 residence time and almost eliminated the transcription delay. Since DNA methylation is widespread within mammalian gene bodies, this suggests a potential mechanism for tuning the kinetic barriers encountered by an elongating RNAP. 9/

Monte Carlo simulations incorporating the measured changes in Egr-1 kinetics reproduced passage through one TF orientation. In the other, they implicated an additional effect on RNAP backtracking or recovery. 8/

Instead, RNAP changed the *kinetic stability* of the Egr-1–DNA complex. When RNAP was within ~20 bp of the binding site, Egr-1 dissociated faster and associated more slowly. Remarkably, these effects varied non-monotonically with RNAP–TF distance. 6/

Could RNAP simply push Egr-1 off the DNA? Force measurements suggest not. The force generated by RNAP was lower than that required to mechanically remove Egr-1, particularly in the more mechanically stable orientation. 5/

Egr-1 delayed RNAP passage, but did not form an absolute block. Importantly, the delay depended on both the orientation of Egr-1 on DNA and the rNTP concentration, arguing against a simple model in which RNAP merely waits for spontaneous TF dissociation. 4/

We developed a non-perturbative assay in which RNAP arrival suppresses fluctuations in a partially unzipped DNA fork. This allowed us to measure transcription through a bound TF without applying force directly to RNAP or labeling either protein. 3/

"Engineering a Mechanoresponsive DNA Origami Capsule for Drug Delivery to Narrowed Arteries" Happy we could contribute to this nice work from Yuval Garini's lab, who also prepared a beautiful cover. pubs.acs.org/doi/10.1021/...

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.