| Summary: | The Escherichia coli melibiose (meý operon encodes proteins that facilitate the metabolism of melibiose. Expression of the mel operon is controlled by a single promoter (the meIAB promoter) and is co-dependent on two transcription activators, MeIR and the cyclic AMP receptor protein (CRP). MeIR is a member of the AraC family of transcription regulators and its activity is controlled by melibiose. CRP is a global regulator controlled by the second messenger, cyclic AMP, whose level is controlled by the availability of glucose. Thus, the co-dependence of the meIAB promoter on two activators couples induction to two physiological signals, the presence of melibiose and the absence of glucose. in the absence of melibiose, MeIR binds to three DNA target sites upstream of the melAB transcription start site. In the presence of melibiose, MeIR is able to bind to a fourth site, which has a low affinity for MeIR and overlaps the -35 hexamer of the melAB promoter. The four MeIR binding sites are organised as two pairs that are separated from each other by a binding site for CRP. Binding of CRP to this site requires co-operative interactions with adjacently bound MeR. These interactions stabilise the binding of both MeIR and CRP to their DNA target sites. In this work it is shown that MeIR binds to each pair of sites at the melAB promoter as a direct repeat. This defines the face of MeIR that is able to interact with adjacently bound CRP and, for MeIR bound close to the -35 hexamer, the a7O subunit of RNA polymerase. The MeIR-e interface has been characterised using a combination of genetics, biochemistry and structural modelling. Amino acid side chains in MeIR and RNA polymeraseth at interact during transcription activation haveb eeni dentified. Finally, simplified derivatives of the melAB promoter, which carry an improved pair of promoter proximal MeIR binding sites, have been used to study the interaction between MeIR and the C-terminal domain of the (x subunit of RNA polymerase. My data show that subunits of MeIR and (X protein can bind to the DNA co-operatively and that this co-operative binding is sufficient to stimulate transcription initiation by RNA polymerase lacking the entire (X C-terminal domain.
|
|---|
