INTERACTION OF GAL REPRESSOR WITH INDUCER AND OPERATOR - INDUCTION OFGAL TRANSCRIPTION FROM REPRESSOR-BOUND DNA

Gal repressor inhibits transcription from the gal promoter (PI) when i t binds to the cognate operator (O-E) The repression is relieved by th e presence of the inducer D-galactose, Compared with its Interaction w ith free repressor, D-galactose binds to the repressor-operator comple x with Ill-fold reduced affinity as determined by fluorescence enhance ment 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 for mation of the inducer-repressor-operator ternary complex has been conf irmed by CD spectral analysis, Fluorescence spectroscopy and energy tr ansfer experiments suggest that individual allosteric effects of the t wo 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-regressor and repressor-operator complexe s, In vitro transcription results demonstrated full derepression of tr anscription of the P1 promoter under conditions In which the concentra tions of the Inducer-repressor binary complex are severalfold higher t han the dissociation constant of the inducer-repressor-operator ternar y complex into inducer-repressor and free DNA, These results strongly suggest that the inducer binding to the repressor-operator complex doe s not lead to dissociation of the repressor from the operator during t ranscription induction, Because Gal repressor inhibits transcription b y modulating the alpha subunit of the P1-bound RNA polymerase, we conc lude that the inducer binding to the operator-bound repressor only all osterically relieves the inhibitory effect of repressor on RNA polymer ase without dissociating the repressor from DNA.