INTERACTIONS BETWEEN GALACTOSE REPRESSOR PROTEIN (GALR) AND THE ALPHA SUBUNIT OF RNA POLYMERASE: REPRESSION AND ACTIVATION OF THE gal PROMOTERS

Abstract

To study molecular mechanisms of gene regulation at the level of transcription, the gal operon of Escherichia coli was used as a model system. Two overlapping promoters P1 and P2 separated by 5 bp and located on opposite faces of the DNA double helix control transcription of galETKM. DNA templates for in vitro transcription experiments were constructed in order to study protein-protein interactions between the repressor GalR, binding at the external operator OE, and the C-terminal domain portion of the alpha subunit of RNA polymerase (α-CTD) binding at gal promoters under non-looping conditions. Partial repression mediated by GalR was quantitated on mutant templates that had only the Pt promoter intact. Activation mediated by GalR was established on mutant templates that had only the P2 promoter intact. Templates were also constructed to alter the geometry between OE and gal promoters. It was shown that GalR mediated repression occurs at either promoter provided GalR binds at OE located on the same face of the DNA double helix as RNA polymerase binding at the promoter, and it was determined that activation occurs at either promoter provided GalR binds at OE located on the opposite face of the DNA with respect to RNA polymerase. Transcription experiments were then performed using mutant RNA polymerases with tryptophan substitutions at sites from amino acids 261 to 270 of α-CTD). It was shown that the same region on α-CTD known to interact with cAMP receptor protein (CRP) at Class I CRP dependent promoters, and with the UP regulatory element at rrnBP, also interacts with GalR when bound at OE, or interacts with DNA near OE, and that it is this interaction that is responsible for GaIR mediated control of transcription. It is proposed that α-CTD may interact with a 3rd DNA regulatory element overlapping OE, and thus stimulate transcription from galP2. Partial repression at the galP1 promoter may be the result of GalR preventing this interaction, by repositioning α-CTD to a site further downstream, to a location where open complex formation is not favored. Alternatively, activation and repression may be due to conformational changes in RNA polymerase induced by protein-protein interactions between α-CTD and GaIR.