Abstract
Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (−) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (−) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (−) supercoils enhance LacI’s DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.
Highlights
Upon binding to the O1, O2, and O3 operators at their native positions? If so, is such trapped superhelicity biologically significant? Here utilizing combined approaches of biochemical assays, bacterial genetics, and atomic force microscopy, we show that upon simultaneously binding the O1 and O2 operators LacI forms a topological barrier that divides a supercoiled plasmid DNA molecule containing the lac promoter region into distinct topological domains
We demonstrate that DNA supercoiling is an important modulator of the basal level of gene expression from the lac operon and that the LacI-mediated topological barrier plays an essential role in this process
Even in the presence of Isopropyl β -D-1-thiogalactopyranoside (IPTG), LacI was able to form multiple LacI-lac O1 complexes and block supercoil diffusion (Table 1 and Supplementary Fig. S3d–g). These results suggest that IPTG is not capable of dissociating LacI from the tandem copies of lac O1 operators, which is consistent with previously published results[26]
Summary
Upon binding to the O1, O2, and O3 operators at their native positions? If so, is such trapped superhelicity biologically significant? Here utilizing combined approaches of biochemical assays, bacterial genetics, and atomic force microscopy, we show that upon simultaneously binding the O1 and O2 operators LacI forms a topological barrier that divides a supercoiled plasmid DNA molecule containing the lac promoter region into distinct topological domains. Upon binding to the O1, O2, and O3 operators at their native positions? If so, is such trapped superhelicity biologically significant? Utilizing combined approaches of biochemical assays, bacterial genetics, and atomic force microscopy, we show that upon simultaneously binding the O1 and O2 operators LacI forms a topological barrier that divides a supercoiled plasmid DNA molecule containing the lac promoter region into distinct topological domains. We demonstrate that DNA supercoiling is an important modulator of the basal level of gene expression from the lac operon and that the LacI-mediated topological barrier plays an essential role in this process
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