Interaction of Gal repressor with inducer and operator: induction of gal transcription from repressor-bound DNA.
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.
Chatterjee
S
S
Department of Biophysics, Bose Institute, Calcutta, India.
Zhou
Y N
YN
Roy
S
S
Adhya
S
S
D002942:Circular Dichroism; D005454:Fluorescence Polarization; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014158:Transcription, Genetic
D012097:Repressor Proteins