| | |  | | | | Record from Web of Science® | | | | | | |
| INTERACTION OF THE DNA-BINDING DOMAIN OF DROSOPHILA HEAT-SHOCK FACTOR WITH ITS COGNATE DNA SITE - A THERMODYNAMIC ANALYSIS USING ANALYTICAL ULTRACENTRIFUGATION |
|
|
| Author(s): KIM SJ, TSUKIYAMA T, LEWIS MS, WU C |
| Source: PROTEIN SCIENCE Volume: 3 Issue: 7 Pages: 1040-1051 Published: JUL 1994 |
| Times Cited: 37 References: 40 |
| Abstract: Heat shock transcription factor (HSF) mediates the activation of heat shock genes by binding to its cognate sites with high affinity and specificity. The high-affinity binding of HSF is dependent on the formation of an HSF homotrimer, which interacts specifically with the heat shock response element (HSE), comprised of 3 inverted repeats of the 5-bp sequence NGAAN. In order to investigate the thermodynamic basis of the interaction between HSF and HSE, we have overexpressed and purified a polypeptide (dHSF(33-163)) encompassing only the DNA-binding domain of HSF from Drosophila and analyzed its binding to DNA by equilibrium analytical ultracentrifugation using a multiwavelength scan technique. We demonstrate that dHSF(33-163) can bind as a monomer with 1:1 stoichiometry to a synthetic 13-bp DNA containing a single NGAAN sequence. The values of the thermodynamic parameters obtained from the temperature dependence of the equilibrium binding constants indicate that the changes of free energy for the binding of dHSF(33-163) to the wild-type site and a mutant DNA site are predominantly characterized by substantial negative changes of enthalpy. Binding to the wild-type DNA is characterized by a significant positive change of entropy, whereas binding to the mutant DNA is distinguished by a negative change of entropy of comparable magnitude. The binding to the mutant DNA was also highly sensitive to increasing salt concentrations, indicating a dominance of ionic interactions. The sequence-specific, 1:1 binding of dHSF(33-163) to the NGAAN sequence provides a basis for the analysis of higher order interactions between HSF trimers and the HSE. |
| Document Type: Article |
| Language: English |
Addresses:
1. NIH, NATL CTR RES RESOURCES, BIOMED ENGN & INSTRUMENTAT PROGRAM, BETHESDA, MD 20892 USA
2. NCI, BIOCHEM LAB, BETHESDA, MD 20892 USA |
| Publisher: CAMBRIDGE UNIV PRESS, 40 WEST 20TH STREET, NEW YORK, NY 10011-4211 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: NU550 |
| ISSN: 0961-8368 |
|
| | | | | | | | | Record from Web of Science® | | | | | | | | | |