Single Molecule Measurements Of The Role Of Tetramer Opening In LacI-mediated DNA Looping

Abstract

Lac repressor (LacI) controls transcription of the genes involved in lactose metabolism. A key role in LacI function is played by its ability to bind simultaneously to two operators, forming a loop in the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested the possibility for the LacI tetramer to adopt different structural conformations by flexing about its C-terminal tetramerization domain. At present it remains unclear to what extent different looping geometries are due to DNA binding topologies rather than distinct protein conformations. We address these questions by employing single molecule tethered particle motion on LacI mutants with intratetramer crosslinking at different positions along the cleft between the two dimers. Measurements on wild-type LacI reveal the existence of three distinct levels of effective tether length, most likely due to the presence of two different DNA looped structures. Restricting conformational flexibility with protein by cross-linking induces clear changes in the tether length distributions, indicating profound effects of tetramer opening (and its limitation due to crosslinking) on the looping conformations available to the system. Our data suggest an important role for large-scale conformational changes of LacI in the looping structures and dynamics.