Tamara A. Belyaeva

A simple mechanism for co-dependence on two activators at an Escherichia coli promoter.

The Escherichia coli melAB promoter is co‐dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co‐operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co‐dependence. MelR binds to four sites, centred at positions −42.5, −62.5, −100.5 and −120.5 relative to the melAB transcription start point. When MelR is pre‐bound, CRP is able to bind to a target located between MelR at positions −62.5 and −100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP‐dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase α subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co‐operatively to adjacent sites and form a bacterial enhanceosome.

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.

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and −10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and −10 region. Parallels with the Escherichia coli araC–araBAD regulatory region are discussed.

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.

Binding of the Escherichia coli MelR protein to the melAB promoter: orientation of MelR subunits and investigation of MelR–DNA contacts

The  Escherichia  coli  MelR  protein  is  a  melibiose‐triggered transcription factor, belonging to the AraC family, that activates transcription initiation at the melAB promoter. Activation is dependent on the binding of MelR to four 18 bp sites, centred at position −42.5 (site 2′), position −62.5 (site 2), position −100.5 (site 1) and position −120.5 (site 1′) relative to the melAB transcription start point. Activation also depends on the binding of CRP to a single site located between MelR binding site 1 and site 2. All members of the AraC family contain two helix–turn–helix (HTH) motifs that contact two segments of the DNA major groove at target sites on the same DNA face. In this work, we have studied the binding of MelR to different sites at the melAB promoter, focusing on the orientation of binding of the two MelR HTH motifs, and the juxtaposition of the different bound MelR subunits with respect to each other. To do this, MelR was engineered to contain a single cysteine residue adjacent to either one or the other HTH motif. The MelR derivatives were purified, and the cysteine residues were tagged with p‐bromoacetamidobenzyl‐EDTA‐Fe, an inorganic DNA cleavage reagent. Patterns of DNA cleavage after MelR binding were then used to determine the positions of the two HTH motifs at target sites. In order to simplify our analysis, we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2′, in the absence of CRP, is sufficient for transcription activation. To assist in the interpretation of our results, we also used a shortened derivative of MelR, MelR173, that is able to bind to site 2 but not to site 2′. Our results show that MelR binds as a direct repeat to site 2 and site 2′ with the C‐terminal HTH located towards the promoter‐proximal end of each site. The orientation in which MelR binds to site 2′ appears to be determined by MelR–MelR interactions rather than by MelR–DNA interactions. In complementary experiments, we used genetic analysis to investigate the importance of different residues in the two HTH motifs of MelR. Epistasis experiments provided evidence that supports the proposed orientation of binding of MelR at its target site.

Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C‐terminal domain of the RNA polymerase α subunit

We have investigated the role of the RNA polymerase α subunit during MelR‐dependent activation of transcription at the Escherichia coli melAB promoter. To do this, we used a simplified melAB promoter derivative that is dependent on MelR binding at two 18 bp sites, located from position −34 to −51 and from position −54 to −71, upstream of the transcription start site. Results from experiments with hydroxyl radical footprinting, and with RNA polymerase, carrying α subunits that were tagged with a chemical nuclease, show that the C‐terminal domains of the RNA polymerase α subunits are located near position −52 and near position −72 during transcription activation. We demonstrate that the C‐terminal domain of the RNA polymerase α subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase α subunit can interact with MelR. Finally, we used alanine scanning to identify determinants in the C‐terminal domain of the RNA polymerase α subunit that are important for MelR‐dependent activation of the melAB promoter.

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.

Bacterial Gene Regulatory Proteins: Organisation and Mechanism of Action

The study of adaptive responses in bacteria led to the discovery of a panoply of proteins whose role is to modulate promoter activity, coupling the expression of specific genes to changes in the environment. These gene regulatory proteins are ubiquitous and are essential for all processes of adaptation and differentiation in all living cells. At first sight, this is a very complex topic, made incomprehensible by the sheer number of different factors and different modes of operation. The aim of this chapter is to tackle the central question of how gene regulatory proteins function, and to describe a small number of key examples from the microbial repertoire that reveal principles applicable to most systems.