Lactose Repressor Functions as a DNA Topological Barrier in Escherichia Coli Lactose Operon

Abstract

Escherichia coli lac repressor (LacI) is a paradigm transcriptional factor that controls the expression of three genes in the lac operon. It is a tetrameric protein, specifically binds to lac operators, such as O1, O2 and O3, and forms a DNA loop to negatively control transcription initiation. Previously, we found that LacI upon binding to multiple lacO1 operators is capable of acting as a DNA topological barrier to block DNA supercoil diffusion and dividing a supercoiled DNA molecule into two independent topological domains (Leng et al. (2011) Proc Natl Acad Sci USA 108: 19973-78). In this study, we showed that LacI is able to function as a topological barrier and block supercoil diffusion upon binding to O1, O2, and O3 operators in the lac operon. Specifically, we demonstrated that LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 operators. Additionally, We carried out time course studies to determine the stability of the topological barriers that are produced by the different LacI-operator complexes. Our results showed that the stability of the topological barriers correlates with the DNA-binding affinity of LacI to the different operators i.e., O1, O2, O3, and Os operators. Furthermore, we confirmed our previous observation in which LacI is able to “keep” certain superhelical energy to stabilize LacI-lacO1 complexes. Our results can be explained by a model in which LacI behaves as a topological barrier in the lac operon to regulate the expression of lacZYA genes in Escherichia coli cells.