Transcriptional activation by natural and artificial cAMP receptor proteins in Escherichia coli

The experiments described in this thesis were performed to analyze complex interactions at prokaryotic promoters controlled by multiple identical activators or through unusual promoter architecture (like protein looping by bivalent DNA-binding proteins). By controlling a promoter with multiple regulator molecules, an organism can integrate many different signals simultaneously, responding to a broad array of effectors. Chapter II details experiments done to determine the mechanism allowing two dimers of the Escherichia coli activator cyclic-AMP Receptor Protein (CRP) to activate transcription from an artificial promoter bearing two CRP-recognition sites. To address this question, we developed a novel technique using directed homodimers to study the individual contributions of homodimeric activators at promoters regulated by more than one molecule of the same activator. We show that at an artificial promoter containing two CRP binding sites at Class I positions both CRP dimers use Activation Region I on their promoter-proximal subunits to interact with RNA polymerase and activate transcription. In Chapter III, we examine a chimeric protein containing two DNA-binding domains, and present evidence that this chimera simultaneously binds to two DNA-recognition sites, forming a DNA-protein loop.