Sequence specificity in DNA binding is mainly governed by association

Abstract

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.

Short tandem repeats bind transcription factors to tune eukaryotic gene expression

“…4E, S72-82; see Supplementary Methods ). Consistent with recent work on E. coli LacI binding to various operator sequences ( 82 ), mean microscopic dissociation rates ( k off, µ ) for sequences with a consensus E-box or a weak E-box were similar but affinities and microscopic association probabilities differed by 12-or 16-fold ( Fig . S82 ).…”

“…These results contradict prior microfluidic measurements suggesting that DNA sequence variation primarily impacts dissociation rates; however, we note that prior experiments did not include dark competitor and therefore likely observed a convolved process of dissociation and rebinding (101,102). Thus, we join other recent work in challenging the canonical view that protein-nucleic acid binding affinities are primarily determined by dissociation rates (82). Our measurements can be explained by a simple 4-state model that suggests STRs enhance affinities by increasing the rate of DNA association, in line with prior work suggesting that degenerate recognition sites may serve as "DNA antennae" to attract TFs to a particular regulatory site (83,(103)(104)(105).…”

Weak tension accelerates hybridization and dehybridization of short oligonucleotides

“…Notably, the chance that oligos separate from one another remains low even after all bases have melted: according to the final strand separation step, a newly-melted duplex is expected to re-form a base pair with p ∼ 0.9 probability. This fast reassociation between short oligos is similar to that recently observed between DNA and the lac repressor [76]. This result also suggests that the unbinding transition state happens after all base pairs have already melted.…”

Transcription factors perform a 2-step search of the nucleus

“…Protein clouds can nucleate anywhere, but they preferentially accumulate at genomic sites with many transcription factor binding sites. Traditionally, the thermodynamic framework assumed constant microscopic on-rates and slower off-rates at cognate sites, but there is accumulating evidence that DNA sequence modulates transcription factor-DNA on rates (Marklund et al 2022). Second, I assume a key feature of transcriptional regulation is enhancer occupancy, or the total fraction of time an enhancer is bound by transcription factors (and not the residence times of individual molecules, which are generally less than 15 seconds) (Sherman and Cohen 2012;Stormo 2013;Chen et al 2014Chen et al , 2021Hansen et al 2018).…”