Single-molecule spectroscopic determination of lac repressor-DNA loop conformation.
The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact "closed" form with a V-shaped repressor and also a more "open" form with an extended hinge. The "9C14" construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.
Morgan
Michael A
MA
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Okamoto
Kenji
K
Kahn
Jason D
JD
English
Douglas S
DS
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D056968:Lac Repressors; D009690:Nucleic Acid Conformation; D012097:Repressor Proteins / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D056968:Lac Repressors; D012097:Repressor Proteins