Interaction of the Escherichia coli Gal repressor protein with its DNA operators in vitro

Abstract

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) 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 degrees 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, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI 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, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered

“…In this paper we have shown that IclR protein is susceptible to various kinds of proteolytic degradation during purification and have established a method of preparing useful amounts of the intact, full-length molecule. The full-length preparations bind to operator DNA with only moderate affinity, showing a half-maximal binding at 3.6 x 10"O M protein, in comparison with other well-characterized repressors where the Kd values fall in the range 10"o-10"2 M (e.g., Vershon et al, 1987;Carey, 1988;Brenowitz et al, 1990). IclR binding of operator is 44-fold weaker when the N-terminal eight or nine amino acids are missing, suggesting that this region contributes significantly to DNA binding.…”

Histone‐like protein HU as a specific transcriptional regulator: co‐factor role in repression of gal transcription by GAL repressor

“…Similar DNA looping by LacI has also been shown with synthetic DNA templates carrying two lac operators (Krämmer et al 1987;Eismann & Müller-Hill 1990). The fact that, in a purified system, the dimeric GalR is not as effective as tetrameric LacI in bringing about gal repression warranted the search for a factor that assists GalR in mediating coordinated repression of both P1 and P2, presumably via DNA looping (Brenowitz et al 1990;Choy & Adhya 1992). This paper shows the isolation of such a factor as the histone-like protein HU.…”

Control of gal transcription through DNA looping: inhibition of the initial transcribing complex.

“…3B), which implies that the repressor inhibits the first phosphodiester bond formation or a step prior to that-i.e., the formation or the activity of the initial transcribing complex (20 (13,16,25). Under the conditions used in our transcription assays, the corresponding operators are fully cpied byl I"I and GaIR+ (16,24).…”