TY - JOUR
T1 - Quantitative DNase Footprint Titration
T2 - A Method for Studying Protein-DNA Interactions
AU - Brenowitz, Michael
AU - Senear, Donald F.
AU - Shea, Madeline A.
AU - Ackers, Gary K.
N1 - Funding Information:
We are very grateful to Ben Turner for his assistance and advice on virtually all aspects of this project. Much of the integration of densities of autoradiograms was performed by Kevin Bootes. We thank Marc Wold and Hillary Nelson for obtaining for us the plasmids used in this study and for many helpful suggestions. We thank Joan Sarkin for patiently word-processing the manuscript. This work was supported by NIH Grant GM 24486. The P-1700 film scanner is an NIH-Facility instrument available to us at The John Hopkins University Medical School.
PY - 1986/1/1
Y1 - 1986/1/1
N2 - This chapter discusses that individual-site binding isotherms are uniquely suited to permit the resolution of interaction parameters for systems exhibiting cooperative interactions between multiple sites. The analysis is completely general. Any number of specific sites can be analyzed regardless of the nature of the cooperative or anticooperative interactions among them. The development of the footprint titration method, which permits resolution of individual-site isotherms, permits quantitative characterization of systems that act as critical regulators of gene transcription. The thermodynamic parameters that are resolved from footprint titration can be used in two important ways to further the understanding of gene regulation. First, the binding affinities of the various components of a gene regulatory system can be used to deduce the mechanism of the regulation—for example, the successful modeling of the switch from the lysogenic-to-lytic growth stage of the lambda phage. Second, the range of precisely controlled experimental conditions over which the technique is applicable allows to measure other thermodynamic parameters—for example, enthalpies and entropies—to study the roles of the various noncovalent forces of interaction involved in protein-DNA binding and site recognition.
AB - This chapter discusses that individual-site binding isotherms are uniquely suited to permit the resolution of interaction parameters for systems exhibiting cooperative interactions between multiple sites. The analysis is completely general. Any number of specific sites can be analyzed regardless of the nature of the cooperative or anticooperative interactions among them. The development of the footprint titration method, which permits resolution of individual-site isotherms, permits quantitative characterization of systems that act as critical regulators of gene transcription. The thermodynamic parameters that are resolved from footprint titration can be used in two important ways to further the understanding of gene regulation. First, the binding affinities of the various components of a gene regulatory system can be used to deduce the mechanism of the regulation—for example, the successful modeling of the switch from the lysogenic-to-lytic growth stage of the lambda phage. Second, the range of precisely controlled experimental conditions over which the technique is applicable allows to measure other thermodynamic parameters—for example, enthalpies and entropies—to study the roles of the various noncovalent forces of interaction involved in protein-DNA binding and site recognition.
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U2 - 10.1016/0076-6879(86)30011-9
DO - 10.1016/0076-6879(86)30011-9
M3 - Article
C2 - 3773731
AN - SCOPUS:0022437260
SN - 0076-6879
VL - 130
SP - 132
EP - 181
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -