Mechanism of promoter repression by Lac repressor–DNA loops

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905, USA.
Nucleic Acids Research (Impact Factor: 9.11). 01/2013; 41(1):156–166. DOI: 10.1093/nar/gks1011


The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repres-sor concentration come from free repressor and re-pressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.

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    • "These findings suggest that repression is poor because of the presence of only one lac operator in the constructs. Because the E. coli lac operon contains three repressor binding sites and because there are examples where two or three operators had to be introduced into expression vectors for the proper functioning of the LacI-IPTG expression system in heterologous hosts (Grespi et al. 2011; Becker et al. 2013), w e m a d e t w o c o ns t r uc t s w i t h th r e e o pe r at or s : pGUSlacIPA3threeoper and pGUSPA3threeoper (Fig. 2a). The pGUSPA3threeoper plasmid, which did not contain lacI, was used as a control. "
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    • "Further, just as in E. coli, the repression both for the non-induced and the induced conditions decrease with longer inter-lacO distances in Synechocystis. This has been shown before in E. coli and illustrates the drop in contribution to the local concentration of LacI at the primary proximal repression site from the distal site for longer distances [23,55]. On the other hand, it was recently demonstrated that the LacI-mediated DNA-loop itself is enough to repress transcription from a T7 promoter positioned inside the loop [55]. "
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