Two amino acid residues from the DNA-binding domain of MalT play a crucial role in transcriptional activation.

MalT is the transcriptional activator of the Escherichia coli maltose regulon. Several lines of evidence suggest that MalT might act by interacting with RNA polymerase. Here, we show that 'MalT, the DNA-binding domain of MalT, activates transcription. In order to identify amino acids of 'MalT playing a specific role in activation, and therefore possibly involved in the putative contact(s) with RNA polymerase, we developed a double screen to isolate mutations of the 'malT gene affecting activation by 'MalT without impairing its DNA-binding affinity. The effect of the mutations thus obtained on activation was assessed in vivo. This strategy essentially pointed to serine 834 and glutamine 876 of the MalT amino acid sequence as specifically involved in activation. Various 'MalT derivatives substituted at positions 834 or 876 were purified and tested in vitro for their DNA-binding affinity, as well as for their activation ability. Together, the results obtained clearly show that serine 834 and glutamine 876 are important for activation by 'MalT but not for DNA-binding. We argue that these amino acid residues are possibly solvent-exposed and propose that they act by contacting RNA polymerase.

Multiple protein-DNA and protein-protein interactions are involved in transcriptional activation by MalT.

The promoters of the Escherichia coli maltose regulon are positively regulated by the MalT protein, which recognizes a short asymmetric nucleotide sequence that is present as several copies in each promoter of the regulon. We report a detailed biochemical characterization of the interaction of MalT with the promoter of the malPQ operon. The MalT sites in malPp were precisely located and their occupation as a function of MalT concentration was quantified using DNase I and dimethyl sulphate footprinting experiments. The contribution of each site to malPp activity was assessed by introducing mutations that destroy them and measuring the residual in vivo and in vitro activity. Two main results were obtained. First, although the proximal MalT site is centred at -37.5, RNA polymerase is likely to establish a contact required for malPp activity with at least one base pair of the promoter -35 region; this close proximity between RNA polymerase and MalT bound to site 1 suggests that the two proteins interact. Second, even if the interaction of MalT with the three functional sites in malPp is a co-operative process, the MalT molecules bound to the two distal sites play a more subtle role than simply increasing the occupancy of the proximal site and may also contact RNA polymerase. We suggest that, in the nucleoprotein structure responsible for the initiation of transcription, MalT, RNA polymerase and malPp are held together by several weak interactions.

Which nucleotides in the "-10" region are crucial to obtain a fully active MalT-dependent promoter?

We have replaced the hexanucleotide corresponding to the "-10" region of malPp, a positively regulated promoter from Escherichia coli, by random nucleotide sequences and isolated 48 different variants that were as active as the wild-type promoter. Analysis of the nucleotide sequence of their "-10" region strongly suggests that the nature of the nucleotide present at three positions plays a crucial role: 46 of the 48 malPp variants contained C or T at position -12, A at position -11 and T at position -7. The nucleotide composition at the three other positions seems to be much more flexible. The features of these "-10" regions are similar to those of the constitutive promoters, but some significant differences are noticeable and will be discussed. In contrast to other positively regulated promoters, none of the various "-10" regions, including the consensus sequence TATAAT, led to a constitutive promoter.

On the puzzling arrangement of the asymmetric MalT-binding sites in the MalT-dependent promoters.

The MalT-dependent promoters of the enterobacteria belong to a small family of positively regulated prokaryotic promoters in which the activator protein recognizes short asymmetric nucleotide sequences present in several locations and orientations. We demonstrate that active MalT-dependent semisynthetic promoters can be constructed by using a synthetic decanucleotide as the MalT site and random nucleotide sequences as connecting sequences, provided that the location and orientation of the sites are the same as in natural MalT-dependent promoters. Strikingly, the induced level of promoter activity and the induction factor of each semisynthetic promoter are identical to those of its natural counterpart, in spite of considerable differences in their nucleotide sequences. The study of these semisynthetic promoters confirms the importance of the structural motif formed by two MalT sites in a direct repeat. This motif is involved in promoter activation either alone or in conjunction with a third MalT site, proximal with respect to the transcription start site. In this latter configuration, the promoters are active irrespective of the orientation of the repeat, and they retain at least some activity when the distance between the repeat and the proximal site is increased, provided that the alignment along the axis of the helix is conserved.

A small C-terminal region of the Escherichia coli MalT protein contains the DNA-binding domain.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, is a 901-amino acid-long protein that specifically binds to short, asymmetric nucleotide sequences present in several copies in the promoters of the regulon. We report that the protein structure involved in this specific binding is carried by a small C-terminal part of MalT encompassing the last 95 amino acid residues. This was demonstrated by fusing the last 95 codons of malT to the gene that encodes glutathione S-transferase, purifying the hybrid protein by affinity chromatography, and comparing the DNase I and dimethyl sulfate footprints of the hybrid and of wild-type MalT on different MalT-binding sites. MalT belongs to a large family of prokaryotic transcriptional activators, which share significant homology in their approximately 60-amino acid C-terminal regions. Our result strongly supports the suggestion that the region of homology corresponds to the DNA-binding domain of the proteins in this family.

Genetic studies on the promoter of malT, the gene that encodes the activator of the Escherichia coli maltose regulon.

We report the construction of a chromosomal malT-lacZ gene fusion that is expressed under the control of the malT promoter in Escherichia coli K12. The resulting hybrid protein is soluble and stable in crude cellular extracts, which allowed us to measure very low levels of malTp activity. In this note, we confirm and extend previous observations on the regulation of malTp. We show that the promoter is 40-times less active in the absence of cAMP receptor protein (CRP) than in its presence, that CRP works by binding to the site centred at position -70.5, and that all of the elements necessary and sufficient for the regulation by CRP are located downstream from position -122.

A new mechanism for coactivation of transcription initiation: repositioning of an activator triggered by the binding of a second activator.

The cAMP receptor protein (CRP) and MaIT, the maltose regulon activator, synergistically activate transcription from the E. coli maIKp promoter. The maIKp regulatory region comprises two series of MaIT-binding sites separated by three CRP-binding sites. By combining genetic and biochemical studies, we demonstrate that the promoter-proximal region contains two overlapping sets of three MaIT-binding sites. Occupation of the higher affinity set of sites, which occurs in the absence of CRP, does not lead to malKp activation. In contrast, in the presence of CRP, MalT binds to the lower affinity set of sites and triggers transcription initiation because, unlike the high affinity set, the low affinity set of sites is properly positioned with respect to the Pribnow box. The CRP effect requires the malKp-distal MalT-binding sites. The synergistic action of MalT and CRP therefore relies on MalT repositioning via the formation of a nucleoprotein structure involving the entire regulatory region.

Supercoiling is essential for the formation and stability of the initiation complex at the divergent malEp and malKp promoters.

malEp and malKp are divergent and partially overlapping promoters of the Escherichia coli maltose regulon, whose activity depends on the presence of two transcriptional activators. MalT and CRP (cAMP receptor protein). Their activation involves a common 210 base-pair regulatory region encompassing multiple binding sites for both activators. Using a supercoiled plasmid containing malEp and malKp as template, purified proteins and a single-round transcription assay, we developed an in vitro system in which both promoters behave as in vivo. In this system, malEp and malKp are active only in the presence of both MalT and CRP, and various mutations in the MalT or CRP binding sites affect the promoters in the same way as they do in vivo. We showed that supercoiling plays a crucial role not only for the formation of the initiation complex at malEp and malKp but also for its stability. In addition, dimethylsulphate protection experiments provide evidence that the nucleoprotein complexes formed by CRP and MalT bound to malEp and malKp on supercoiled and relaxed DNA are different. We speculate that one of the roles of supercoiling might be to assist the assembly of a preinitiation complex involving the regulatory region DNA and several molecules of MalT and CRP.

Two MalT binding sites in direct repeat. A structural motif involved in the activation of all the promoters of the maltose regulons in Escherichia coli and Klebsiella pneumoniae.

The maltose regulons of Escherichia coli and Klebsiella pneumoniae are very similar, comprising three operons that code for the proteins required for the utilization of maltodextrins as a carbon source. The maltose regulon of K. pneumoniae contains two additional operons, pulAB and pulC-O, which allow the use of starch as a carbon source. The promoters of all of these operons are strictly controlled by the activator protein MalT. In this paper, we report a detailed study of the structure and the functional role of the MalT binding sites located in the adjacent and divergent pulAp and pulCp promoters. By biochemical and genetic experiments, we show that the 134 base-pair region separating the transcription start sites of pulAp and pulCp contains four MalT binding sites, which leads us to propose a revised consensus for the asymmetrical nucleotide sequence recognized by MalT (5'-GGGGAT/GGAGG). MalT binds co-operatively to these four sites, contacting the major groove of the DNA helix. The genetic dissection of the pulAp-pulCp region shows that the promoters partially overlap: the two central MalT binding sites, which are in direct repeat, are required for the activation of both promoters. We further show that an analogous pair of directly repeated MalT binding sites is also involved in the activation of two other promoters of the regulon, malEp and malKp. This study, which confirms the striking structural diversity of the promoters of the maltose regulon, suggests that the motif formed by two MalT binding sites in direct repeat is a recurrent feature of these promoters and plays a crucial role in their activation.

Three adjacent binding sites for cAMP receptor protein are involved in the activation of the divergent malEp-malKp promoters.

The divergent malEFG and malK-lamB-malM operons in Escherichia coli are controlled by partially overlapping promoters, whose activity depends on the presence of two transcriptional activators, MalT and the cAMP receptor protein (CRP). The 271-base-pair region separating the transcription start points of the promoters malEp and malKp comprises a compact array of binding sites for MalT and CRP. We report the characterization of the in vitro interactions of CRP with its four adjacent binding sites and the analysis of their function in vivo. By using the DNase I footprinting technique, we showed that CRP binds with high affinity to the three malEp-proximal sites and with a low affinity to the fourth site. CRP binding to these sites is not cooperative, even though they are adjacent and located on the same face of the DNA double helix. Each of these sites was destroyed by localized mutagenesis and the residual activity of the promoters was measured in vivo. Mutations in any of the three high-affinity binding sites reduced both malEp and malKp activity. The participation of several adjacent bound CRP molecules in the activation of a promoter is an unprecedented observation and might involve molecular mechanisms quite different from those used in the other CRP-controlled promoters.

Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation.

In Escherichia coli transcription of individual genes generally requires concomitant translation, and thus the decay of mRNAs cannot be studied without the complication of translation. Here we have used T7 RNA polymerase to transcribe in vivo lacZ genes carrying ribosome binding sites of variable efficiency. We show that neither cell viability nor growth rate is affected by the T7-driven transcription of these genes, provided that they are present as single chromosomal copy. Furthermore, transcription is now completely uncoupled from translation, allowing large amounts of even completely untranslated mRNAs to be synthesized. Taking advantage of these features, we discuss the influence of the frequency of translation upon the processing and degradation of the lac message.

Characterization of malT mutants that constitutively activate the maltose regulon of Escherichia coli.

The expression of the maltose regulon of Escherichia coli is controlled by a transcriptional activator, the product of the malT gene, and is induced by the presence of maltose or maltodextrins in the growth medium. We isolated eight mutants with mutations in malT which lead to constitutive expression of the regulon. The nucleotide sequences of the mutated genes revealed that the eight mutations are clustered in two small regions in the first one-third of the malT gene. Two mutated MalT proteins (corresponding to a mutation in each cluster) were purified and examined for in vitro activation of the MalT-dependent malPp promoter. Whereas wild-type MalT activity was absolutely dependent upon the presence of maltotriose, even at high protein concentrations, both mutated proteins were partially active in the absence of this sugar. Indeed, while the activity of the mutated proteins was still increased by maltotriose at low protein concentrations, the proteins were fully active in the absence of maltotriose at high protein concentrations. Both proteins exhibited a fivefold-higher affinity for maltotriose than the wild-type protein did.

MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator.

We show that MalT, the transcriptional activator of the Escherichia coli maltose regulon, specifically binds ATP and dATP with a high affinity (Kd = 0.4 microM) and exhibits a weak ATPase activity. Using an abortive initiation assay, we further show that activation of open complex formation by MalT depends on the presence of ATP in addition to that of maltotriose, the inducer of the maltose system. Similar experiments in which ATP was replaced by ADP or AMP-PNP, a non-hydrolysable analogue of ATP, demonstrate that this reaction does not require ATP hydrolysis. As revealed by DNase I footprinting, both ATP and maltotriose are required for the binding of the MalT protein to the mal promoter DNA.

Nucleoprotein structures at positively regulated bacterial promoters: homology with replication origins and some hypotheses on the quaternary structure of the activator proteins in these complexes.

The quaternary structure of regulatory proteins undoubtedly plays an important role in the initiation of transcription and DNA replication. To date, the best-characterized regulatory proteins are oligomers in which promoters are bound together by isologous interactions. From the examples presented in this article, it appears that the formation of certain nucleoprotein complexes implicated in transcription initiation might involve heterologous rather than isologous interactions, allowing differentiation between two classes of transcription activators. Nucleoprotein structures present at the oriC replication origin and at malB promoters show striking homologies.

A complex nucleoprotein structure involved in activation of transcription of two divergent Escherichia coli promoters.

Initiation of transcription at malEp and malKp, two divergent Escherichia coli promoters, depends on the presence of both CRP, a pleiotropic activator, and MalT, the maltose regulon activator. We carried out in vivo genetic and functional analysis of these promoters and characterized their interaction with MalT and CRP using DNase I footprinting. The functional limits of the promoters are located about 240 base-pairs (bp) upstream of their transcription start sites, which are 271 bp apart. These promoters therefore overlap by about 210 bp. The overlapping region encompasses four CRP-binding sites and at least four MalT-binding sites. Insertions in the centre of this region are tolerated provided that they correspond to an integral number of DNA helix turns. In DNase I footprinting experiments performed on the complex formed by MalT with malEp-malKp, the DNA appears to be wrapped around the protein. We propose a model for the nucleoprotein structure that might be involved in transcription activation at these divergent promoters.

Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon.

Expression of the Escherichia coli maltose regulon is controlled by MalT, a transcriptional activator (Mr = 102,288) encoded by the malT gene. Activation of transcription depends on the presence of the inducer, maltotriose. Using an in vitro transcription/translation assay to monitor the protein, we have purified MalT in native form from MalT-overproducing bacteria. The purified protein is able to promote transcription from different MalT-controlled promoters in well-defined in vitro systems. Maltotriose and the MalT protein suffice to stimulate initiation of transcription at malPp by the E. coli RNA polymerase holoenzyme. In contrast, both MalT protein and cAMP receptor protein are required with their respective effectors, maltotriose and cyclic AMP, for activation of malEp. These data are in agreement with in vivo observations. In addition, we present evidence that MalT is an ATP-binding protein, a result suggesting that ATP may play a role in transcription initiation.

Maltotriose is the inducer of the maltose regulon of Escherichia coli.

In a cell-free system programmed with a plasmid bearing a malP'-'lacZ gene fusion under the control of malPp, beta-galactosidase synthesis was strictly dependent on the presence of both the MalT activator protein and the inducer of the Escherichia coli maltose regulon. We show that, among all maltodextrins tested (from maltose to maltoheptaose), only maltotriose was able to induce beta-galactosidase synthesis. Likewise, in an in vitro transcription system, initiation of transcription at malPp required the presence of the MalT protein and maltotriose along with the RNA polymerase holoenzyme; neither maltose nor maltotetraose could substitute for maltotriose.

Comparison of the malA regions of Escherichia coli and Klebsiella pneumoniae.

Using the mini-Mu-duction technique, we cloned the malA regions from Escherichia coli K-12 and Klebsiella pneumoniae. A comparison of the structures of the cloned DNAs indicated that the malT, malP, and malQ genes, encoding the transcriptional activator of the maltose regulon, maltodextrin phosphorylase, and amylomaltase, respectively, are similarly organized in both species; malP and malQ constitute an operon divergent from the malT gene. We sequenced 1,200 nucleotides encompassing the beginnings of the malT and malP genes, their promoters, and the intergenic region. The DNA sequences from the two species were very different; the levels of homology ranged from 28 to 80%, depending on the region. The sequences of the coding regions and of elements known to be important for the functions of these two promoters in E. coli were well conserved between the two bacteria, whereas the sequence of the malT-malP intergenic region had totally diverged.

[Preliminary characterization of a new system allowing maltose assimilation by Escherichia coli]. / Caractérisation préliminaire d'un nouveau système permettant l'assimilation du maltose par Escherichia coli.

We showed that Klebsiella pneumoniae and Erwinia herbicola possessed a pathway for maltose metabolism which could function in parallel with that encoded by the classical maltose regulon. Indeed, specific DNA fragments isolated from these two bacteria allowed growth on maltose of any of the mal mutants of Escherichia coli when in a multicopy number. The preliminary characterization of the E. herbicola DNA fragment indicated that a 4-Kb region was sufficient to complement the mal mutations of E. coli, and that this region encoded at least one 50-Kd protein. This protein is probably bound to the cytoplasmic membrane and its synthesis is induced by maltose independently of malT, the positive regulator gene of the maltose regulon.