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

Abstract

DNA looping mediated by the lac Repressor protein (LacI) is a paradigm of study for protein-DNA interaction as well as DNA mechanics on the nanoscale. Loop formation in this system occurs by the spontaneous binding of LacI to two distant operators on its DNA substrate. We employ single-molecule Tethered Particle Motion (TPM) to observe loop formation and breakdown in various DNA substrates. We are discussing and aiming to explain substantial differences between loop lifetime measurements in bulk and single-molecule experiments, namely an unexpected difficulty in competing bound protein off in the presence of excess DNA even though loops continued to form and break down rapidly. For this aim, we conduct single-molecule DNA competition experiments using DNAs with different operator strengths and intra-operator sequences giving rise to intrinsic bends. We find that we are able to compete off LacI that is bound to unbent DNA constructs with non-ideal operators in a matter of minutes, in line with typical loop breakdown rates. In contrast, competing off LacI bound to unbent or intrinsically curved DNA substrates with ideal operators took at least hours up to days in both single-molecule and bulk experiments, even though loops continue to break down and form repeatedly within minutes. To explain this resistance to competition of the LacI-DNA complex in DNA substrates with ideal operators while loops continue to break down, we posit a weak binding of LacI to non-operator DNA in the unlooped state; this hypothesis is supported by recent complementary experiments that reveal interactions between LacI and non-operator DNA outside the looping region.