INTERACTION OF THE ESCHERICHIA-COLI GAL REPRESSOR PROTEIN WITH ITS DNA OPERATORS INVITRO

被引:82
作者
BRENOWITZ, M [1 ]
JAMISON, E [1 ]
MAJUMDAR, A [1 ]
ADHYA, S [1 ]
机构
[1] NCI,MOLEC BIOL LAB,BETHESDA,MD 20892
关键词
D O I
10.1021/bi00465a033
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and O1) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683–9687] has suggested the role of a regulatory “looped complex” mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 °C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, ΔGI and ΔGE, and a constant describing cooperativity, ΔGIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 °C was ΔGIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. ΔGE and ΔGI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, ΔCp, of-2.3 kcal mol‒1 deg−1. Mobility-shift titration experiments conducted at 20 and 0 °C yielded values for ΔGIE that were consistent with those resolved from the footprint analysis. Unique values of ΔGIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that ΔGE = ΔGI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE‒ and OI‒ operators. © 1990, American Chemical Society. All rights reserved.
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页码:3374 / 3383
页数:10
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