Tetramer opening in LacI-mediated DNA looping.
Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations.
Rutkauskas
Danielis
D
European Laboratory for Nonlinear Spectroscopy and Department of Physics, University of Florence, 50019 Sesto Fiorentino (FI), Italy.
Zhan
Hongli
H
Matthews
Kathleen S
KS
Pavone
Francesco S
FS
Vanzi
Francesco
F
D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D056968:Lac Repressors / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D055503:Protein Multimerization
D004269:DNA, Bacterial; D056968:Lac Repressors