DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA.
Escherichia coli HU(alphabeta), a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU(alphabeta) interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (> or =34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1+/-0.4x10(6) M(-1), and a binding enthalpy of +7.7+/-0.6 kcal/mol at 15 degrees C and 0.15 M Na(+). With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (omega approximately 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1+/-0.2x10(5) M(-1) for the 10 bp mode, 3.5+/-1.4x10(4) M(-1) for the 6 bp mode) and binding enthalpies (4.2+/-0.3 kcal/mol for the 10 bp mode, -1.6+/-0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143+/-6 degrees whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.
Koh
Junseock
J
Program in Biophysics, University of Wisconsin, Madison WI 53706, USA.
Saecker
Ruth M
RM
Record
M Thomas
MT
D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009690:Nucleic Acid Conformation; D013696:Temperature; D013816:Thermodynamics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C116822:hns protein, E coli; D004247:DNA