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 repressor concentration
come from free repressor and repressor 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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    ABSTRACT: Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-d-thiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (β-glucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyper-inducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.
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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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    ABSTRACT: Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library). We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis. The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.
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