Christine L. Webster

Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein.

MelR is a melibiose‐triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR‐dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR‐and CRP‐binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position −100.5 (Site 1) and position −62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1′, centred at position −120.5, located immediately upstream of Site 1, and Site R, at position −238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1′, Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2′, centred at position −42.5) located immediately downstream of Site 2, and overlapping the −35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem‐bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2′. Optimal expression of the melAB promoter requires Sites 1′, Site 1, Site 2 and Site 2′; CRP acts as a ‘bridge’ between MelR bound at Sites 1′ and 1 and at Sites 2 and 2′, increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2′ removes the requirement for Site 1′ and Site 1, and short circuits the effects of CRP.

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.

Organisation of the regulatory region of the Escherichia coli melibiose operon

The regulatory region of the Escherichia coli melibiose operon contains two divergent promoters. One promoter is responsible for the expression of the melR gene, that is essential for melibiose-dependent stimulation of the second promoter. Melibiose-induced transcription from this second promoter initiates at a start point 25 bp upstream from the start codon of the melA gene, encoding an alpha-galactosidase. The nucleotide sequence covering the divergent promoters and the melR gene is reported.

Transcription from the Escherichia coli melR promoter is dependent on the cyclic AMP receptor protein

Expression of the melR gene is required for melibiose-dependent stimulation of transcription initiation at the promoter of the melAB operon. Using the S1 nuclease method we have located the melR transcription start point. Transcription from the melR promoter is dependent on cAMP-CRP: specific nucleotide sequences downstream of bp -59 with respect to the melR transcription start are sufficient for full promoter activity. Nucleotide sequence homologies suggest that the cAMP-CRP binding site is located from bp -52 to -31, in exactly the same position as at the galP1 promoter. Using DNase I footprinting we show that cAMP-CRP and RNA polymerase together bind tightly to the melR promoter sequence, creating a strong footprint from bp -70 to +20. Alone, cAMP-CRP binding is hardly detectable, whereas RNA polymerase alone creates a weak footprint centred around the -10 hexamer sequence. When the melR gene is expressed from a cAMP-CRP-independent promoter, melibiose-dependent transcription from the melAB promoter becomes independent of cAMP-CRP, showing that the melR promoter is the primary site of control by cAMP-CRP in the mel regulon.

Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase σ 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 σ. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase σ70 subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of σ70. 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 σ.

The Escherichia coli meIR gene encodes a DNA-binding protein with affinity for specific sequences located in the melibiose-operon regulatory region

Crude extracts, made from Escherichia coli cells carrying a plasmid in which the melR gene was expressed from the galP2 promoter, were used as a source of MelR protein. Using DNase I footprinting and gel retardation assays, we show that MelR binds to two sites located from nucleotides (nt) -49 to -75 and -85 to -113, upstream from the melAB transcription start point. The two sites contain identical 18-bp sequences. Specific binding is unaltered by deletions that remove 1 or 6 amino acids (aa) from the C terminus of MelR, but is abolished by deletion of 16, 24 or more aa residues. Sequence homologies between MelR and other DNA-binding proteins are discussed.

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.

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.