DNA Competition Experiments Reveal the Importance of Operator Binding Strength and Inter-Operator Sequence in Protein-Mediated DNA Looping

Abstract

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 lac-ZYA genes in Escherichia coli cells.