We are studying the mechanisms by which the two promoters of the gal operon of E. coli are regulated. We have previously shown that each of the promoters is negatively regulated by binding of Gal Repressor to two operator elements, one of which (OE) is located upstream to the promoters and the other (OI) inside the galE structural gene. OE and OI are separated by 114 bp. We have proposed various models by which Gal Repressor inhibits transcription by binding to two distal sites. These models have been tested by various genetic and biochemical experiments: competition binding experiments between Repressor, RNA polymerase and CRP; measurement of binding energies when Repressor binds to OE and OI; the effect of changing the angular orientation between the two Repressor contact points; physical measurements of the effect of Repressor binding of DNA structure; changing one or both of the operators into lac operator(s) by site directed mutagenesis and then studying the effect of Gal and Lac Repressors on operator combinations: OGE-OGI, OGE-OLI, OLE-OGI and OLE-OLI. The results obtained so far suggest that Gal Repressor binding inhibits gal expression not by sterically hindering the binding of RNA polymerase and/or CRP, but by changing the structure of RNA polymerase to an inactive form. We are also studying the structure of Gal Repressor and the nature of its interaction with the operators by genetic and biochemical analysis, e.g. mutational analysis and defining the contact points by chemical protection studies. The results show that each half of symmetrical operator is occupied by a subunit of dimeric Gal Repressor. Gal Repressor contacts at least two major grooves lying on one face of the dyad symmetry. A third major groove on the opposite face is also affected by Repressor either through direct contact by wrapping Repressor around the helix, or indirectly by Repressor changing the DNA helical structure.
