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Interaction of Gal repressor with inducer and operator: induction of gal transcription from repressor-bound DNA.

ABSTRACT: Gal repressor inhibits transcription from the gal promoter (P1) when it binds to the cognate operator (O(E)). The repression is relieved by the presence of the inducer D-galactose. Compared with its interaction with free repressor, D-galactose binds to the repressor-operator complex with 10-fold reduced affinity as determined by fluorescence enhancement measurements. Thermodynamic analysis and fluorescence anisotropy showed that the stability of the repressor-operator complex is reduced by only 7-fold by the presence of the inducer in the complex. The formation of the inducer-repressor-operator ternary complex has been confirmed by CD spectral analysis. Fluorescence spectroscopy and energy transfer experiments suggest that individual allosteric effects of the two ligands, inducer and operator, on Gal repressor are responsible for the slightly weakened stability of the ternary complex compared with the stability of the inducer-repressor and repressor-operator complexes. In vitro transcription results demonstrated full derepression of transcription of the P1 promoter under conditions in which the concentrations of the inducer-repressor binary complex are severalfold higher than the dissociation constant of the inducer-repressor-operator ternary complex into inducer-repressor and free DNA. These results strongly suggest that the inducer binding to the repressor-operator complex does not lead to dissociation of the repressor from the operator during transcription induction. Because Gal repressor inhibits transcription by modulating the alpha subunit of the P1-bound RNA polymerase, we conclude that the inducer binding to the operator-bound repressor only allosterically relieves the inhibitory effect of repressor on RNA polymerase without dissociating the repressor from DNA.

SUBMITTER: Chatterjee S

PROVIDER: S-EPMC20304 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Project description:The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.