Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF.
Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from approximately 2 ms(-1) at approximately 37 degrees C to approximately 40 ms(-1) at approximately 10 degrees C and correspond to an activation energy of approximately 14 +/- 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.
Kuznetsov
Serguei V
SV
Department of Physics (M/C 273), University of Illinois, 845 West Taylor Street, Chicago, IL 60607, USA.
Sugimura
Sawako
S
Vivas
Paula
P
Crothers
Donald M
DM
Ansari
Anjum
A
D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D034982:Integration Host Factors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D013816:Thermodynamics
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C440502:integration host factor, E coli