Two DNA sites for MelR in the same orientation are sufficient for optimal MelR-dependent repression at the Escherichia coli melR promoter.

The Escherichia coli melR gene encodes the MelR transcription factor that controls melibiose utilization. Expression of melR is autoregulated by MelR, which represses the melR promoter by binding to a target that overlaps the transcript start. Here, we show that MelR-dependent repression of the melR promoter can be enhanced by the presence of a second single DNA site for MelR located up to 250 base pairs upstream. Parallels with AraC-dependent repression at the araC-araBAD regulatory region and the possibility of the MelR-dependent repression loop formation are discussed. The results show that MelR bound at two distal loci can cooperate together in transcriptional repression.

The Escherichia coli regulator of sigma 70 protein, Rsd, can up-regulate some stress-dependent promoters by sequestering sigma 70.

The Escherichia coli Rsd protein forms complexes with the RNA polymerase sigma(70) factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative sigma(38) factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with sigma(70). Our experiments support a model in which the role of Rsd is primarily to sequester sigma(70), thereby increasing the levels of RNA polymerase containing the alternative sigma(38) factor.

Mutational analysis of the Escherichia coli melR gene suggests a two-state concerted model to explain transcriptional activation and repression in the melibiose operon.

Transcription of the Escherichia coli melAB operon is regulated by the MelR protein, an AraC family member whose activity is modulated by the binding of melibiose. In the absence of melibiose, MelR is unable to activate the melAB promoter but autoregulates its own expression by repressing the melR promoter. Melibiose triggers MelR-dependent activation of the melAB promoter and relieves MelR-dependent repression of the melR promoter. Twenty-nine single amino acid substitutions in MelR that result in partial melibiose-independent activation of the melAB promoter have been identified. Combinations of different substitutions result in almost complete melibiose-independent activation of the melAB promoter. MelR carrying each of the single substitutions is less able to repress the melR promoter, while MelR carrying some combinations of substitutions is completely unable to repress the melR promoter. These results argue that different conformational states of MelR are responsible for activation of the melAB promoter and repression of the melR promoter. Supporting evidence for this is provided by the isolation of substitutions in MelR that block melibiose-dependent activation of the melAB promoter while not changing melibiose-independent repression of the melR promoter. Additional experiments with a bacterial two-hybrid system suggest that interactions between MelR subunits differ according to the two conformational states.

Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit.

Activation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family. MelR binds to 18 bp target sites using two helix-turn-helix (HTH) motifs that are both located in its C-terminal domain. The melAB promoter contains four target sites for MelR. Previously, we showed that occupation of two of these sites, centred at positions -42.5 and -62.5 upstream of the melAB transcription start point, is sufficient for activation. We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned. Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site. As our model for DNA-bound MelR suggests that HTH 2 must be adjacent to the melAB promoter -35 element, we searched this part of MelR for amino acid side-chains that might be able to interact with sigma. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase sigma(70) subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of sigma(70). In the final part of the work, we describe experiments in which the MelR binding site at position -42.5 was improved. We show that this is detrimental to MelR-dependent transcription activation because bound MelR is mispositioned so that it is unable to make 'correct' interactions with sigma.

Architectural requirements for optimal activation by tandem CRP molecules at a class I CRP-dependent promoter.

The Escherichia coli cyclic AMP receptor protein (CRP) activates transcription at target promoters by interacting with the C-terminal domain of the RNA polymerase alpha subunit. We have constructed a set of promoters carrying tandem DNA sites for CRP with one site centred at position -61.5 and the other site located at different upstream positions. Optimal CRP-dependent activation of transcription is observed when the upstream DNA site for CRP is located at position -93.5 or at position -103.5. Evidence is presented to suggest that activation by the upstream-bound CRP molecule is due to interaction with the C-terminal domain of the RNA polymerase alpha subunit.