Using synthetic bacterial enhancers to reveal a looping-based mechanism for quenching-like repression

Abstract

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.

A looping-based model for quenching repression

“…2A. The data show that in the elastic regime (L ≤ b), protrusions bound in-phase with δr (solid lines, ↑) strongly reduce the looping probability relative to that of the bare DNA, while protrusions positioned out-of-phase to δr (dashed lines, ↓) increase the looping probability, as we showed previously (37). However, in the entropic regime (L b), all chains converge to values of F…”

“…Our work indicates that we should consider three ranges for the physics of looping of chromatinized DNA. For short ranges which we studied previously (14,37), looping is dominated by elastic energy. As shown in this work, there is an intermediate entropic looping range of up to ∼10 kbp of bare DNA (or ∼100 kbp of "mixed" DNA).…”

“…The data show that in the elastic regime, the "outside" geometry (green line, K < 0) generates a significantly smaller effect on the probability of looping as compared with the "inside" architecture (blue line, K > 0). This lack of symmetry is probably due to the propensity of short loops to form a "tear-drop" shape, thereby reducing the quenching effect of any elements bound outside the looping segment (37). However, for sufficiently large L/b, F (L) for both ±K enhancer geometries converge to the same value, as predicted by the eclipse model.…”

“…The first N 1 links of the chain were simulated with link length l 1 = 0.34 nm, corresponding to the length of a base-pair in dsDNA. We used ∆i ≈ 4 3 w l for this stage (37). All bound objects were positioned at links k < N 1 .…”

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

“…Studies exploring the formation of this repression loop have found that it is heavily dependent on the spacing of the LacI operator sites relative to each other [28][29][30] . Due to the helical nature of B-form DNA, which completes a full rotation roughly every 10.5 bp, as operator sites are placed at various distances from one another along the DNA this also changes their relative orientation along the face of the DNA helix.…”