Rapid Formation And Breakdown Of Protein-mediated DNA Loops

Abstract

The Lactose Repressor protein (LacI) is a paradigm for the study of transcriptional regulation and protein-DNA interaction. LacI represses transcription of the Lac operon in E. Coli by binding to two distant operator sites and bending the intervening DNA into a DNA loop. Despite a wealth of knowledge on the biochemistry of this process, the details of the binding dynamics are still unresolved, and are the subjects of several lines of current investigations. We present Tethered Particle Microscopy (TPM) data on designed hyperstable loop-forming DNA constructs and find that LacI-mediated DNA loops form and break down on time scales of the order of minutes. This is in stark constrast to measurements in competition assays by Mehta et al1, who report loop lifetimes of days for these constructs. We propose two possible explanations for the LacI-loop formation process that hamronizes these seemingly contradictory observations. Specifically, we propose that the loop-forming LacI tetramer is destabilized by binding to the DNA, and that therefore the primary loop breakdown process is a dissociation of the tetramer into two DNA-bound dimers, which is in contradiction to the prevailing model for this process. Alternatively, we discuss what assumptions have to be made to explain these experimental results purely in terms of dissociation of the tetramer from DNA. Namely, we need to assume an outsized effect of spatial operator orientation on loop formation rates and postulate that the protein is extremely inflexible.1. Mehta, R.A. and Kahn, J.D. “Designed hyperstable lac-Repressor-DNA loop topologies suggest alternative looop geometries.” Journal of Molecular Biology 294 (1999), 67-77.