We explore a model for ‘quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10–11 bp insertions or deletions (INDELs) and sensitive to 5–6 bp INDELs. We test this prediction on 61 σ54-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat.

We compute for the first time the effects of excluded volume on the probability for double-stranded DNA to form a loop. We utilize a Monte-Carlo algorithm for generation of large ensembles of selfavoiding worm-like chains, which are used to compute the J-factor for varying lengthscales. In the entropic regime, we confirm the scaling-theory prediction of a power-law drop off of −1.92, which is significantly stronger than the −1.5 power-law predicted by the non-self-avoiding worm-like chain model. In the elastic regime, we find that the angle-independent end-to-end chain distribution is highly anisotropic. This anisotropy, combined with the excluded volume constraints, lead to an increase in the J-factor of the self-avoiding worm-like chain by about half an order of magnitude relative to its non-self-avoiding counterpart. This increase could partially explain the anomalous results of recent cyclization experiments, in which short dsDNA molecules were found to have an increased propensity to form a loop.

We model the regulatory role of proteins bound to looped DNA using a simulation in which dsDNA is represented as a self-avoiding chain, and proteins as spherical protrusions. We simulate long self-avoiding chains using a sequential importance sampling Monte-Carlo algorithm, and compute the probabilities for chain looping with and without a protrusion. We find that a protrusion near one of the chain's termini reduces the probability of looping, even for chains much longer than the protrusion-chain-terminus distance. This effect increases with protrusion size, and decreases with protrusion-terminus distance. The reduced probability of looping can be explained via an eclipse-like model, which provides a novel inhibitory mechanism. We test the eclipse model on two possible transcription-factor occupancy states of the D. melanogaster eve 3/7 enhancer, and show that it provides a possible explanation for the experimentally-observed eve stripe 3 and 7 expression patterns. Author SummaryBiological regulation-at-a-distance, whereby a transcription factor (TF) is able to generate susbstantial regulatory effects on gene expression even though it may be bound a large distance away from its target (500 bp-1 Mbp), is only partially understood. Using a biophysical model and a computer simulation that take dsDNA and TF volumes into account, we identify a downregulatory mechanism which functions at large distances, whereby a TF bound within * 150 bp from an activator decreases the probability of looping-based interaction between the activator and the distant core promoter. This "eclipse" mechanism provides insight into the question of how enhancer architecture dictates gene expression.

We model the regulatory role of proteins bound to looped DNA using a simulation in which dsDNA is represented as a self-avoiding chain, and proteins as spherical protrusions. We simulate long self-avoiding chains using a sequential importance sampling Monte-Carlo algorithm, and compute the probabilities for chain looping with and without a protrusion. We find that a protrusion near one of the chain’s termini reduces the probability of looping, even for chains much longer than the protrusion–chain-terminus distance. This effect increases with protrusion size, and decreases with protrusion-terminus distance. The reduced probability of looping can be explained via an eclipse-like model, which provides a novel inhibitory mechanism. We test the eclipse model on two possible transcription-factor occupancy states of the D. melanogaster eve 3/7 enhancer, and show that it provides a possible explanation for the experimentally-observed eve stripe 3 and 7 expression patterns.

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