Flanking DNA Matters in DNA Loop Formation and Breakdown Mediated by Lac Repressor Protein

Abstract

The lac Repressor protein (LacI) is a paradigm for the study of protein-mediated DNA loops in bacteria. When it binds to two distant operator sites on substrate DNA, it causes the formation of DNA loops. Although past in vitro and in vivo experiments have shown that changes in the inter-loop DNA can affect loop formation and breakdown, here I will present in vitro experimental evidence that the DNA outside the loop that is flanking the operators can also affect looping kinetics. Observing loop formation and breakdown in single molecules with Tethered Particle Motion (TPM) we found fluctuations in how much DNA is bound to the protein, and these fluctuations depend on the AT- or GC-content of the flanking regions outside the loop. This suggests an interaction between the looped complex and the flanking region which has not been previously observed. The studies complement previous experiments which found that DNA between the operator sites interacts with LacI while in the looped state. These results suggest that the presence or absence of flanking DNA interactions with the protein complex could give rise to additional states in TPM experiments that are not caused by conformational changes of the protein complex or topologic variations of the loop.