Related Experiment Videos
Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor
K Bondeson1, A Frostell-Karlsson, L Fägerstam
1Department of Medical Virology, Uppsala University Biomedical Centre, Sweden.
Analytical Biochemistry
|October 1, 1993
Summary
This study monitored lactose repressor binding to operator DNA using a biosensor. Researchers determined the dissociation rate constant for the lac repressor-DNA complex, providing new insights into gene regulation.
Area of Science:
- Molecular Biology
- Biophysics
- Biochemistry
Background:
- The lactose repressor (lac repressor) controls the transcription of the lac operon in E. coli.
- Understanding the kinetics of lac repressor-operator DNA binding is crucial for comprehending gene regulation.
- Previous studies have characterized the equilibrium binding, but real-time kinetic data, especially dissociation rates, are less understood.
Purpose of the Study:
- To continuously monitor the binding and dissociation of the lactose repressor to operator DNA using a biosensor.
- To determine the association (kass) and dissociation (kdiss) rate constants for the lac repressor-operator complex.
- To compare biosensor results with traditional methods like electrophoretic mobility shift assay (EMSA).
Main Methods:
- Utilized surface plasmon resonance (SPR) biosensor technology to measure real-time interactions.
- Immobilized synthetic double-stranded operator DNA onto the sensor surface.
- Passed purified repressor protein over the immobilized DNA and measured complex formation and dissociation.
- Confirmed specificity using isopropyl-beta-D-thiogalactopyranoside (IPTG) inducer.
- Performed electrophoretic mobility shift assay (EMSA) for comparative analysis.
Main Results:
- Demonstrated specific binding of the lac repressor to the operator DNA, inhibited by IPTG.
- Determined the apparent association rate constant (kass) to be 1.8 x 10^6 M^-1 s^-1.
- For the first time, determined the uncatalyzed dissociation rate constant (kdiss) for the lac repressor-operator complex as 3.4 x 10^-4 s^-1.
- Calculated an equilibrium binding constant from biosensor data of 5.1 x 10^9 M^-1, significantly higher than the 2.4 x 10^8 M^-1 obtained via EMSA.
Conclusions:
- Surface plasmon resonance (SPR) provides a powerful tool for real-time kinetic analysis of protein-DNA interactions.
- The determined kinetic parameters, particularly the dissociation rate, offer a more comprehensive understanding of lac repressor function.
- The discrepancy in equilibrium binding constants highlights the sensitivity and potential advantages of SPR for such analyses.