Biomedical subjects

E Richet

Publications and source records attributed to E Richet.

At least 19 recordsLinked to original sources

PubMed2000

Synergistic transcription activation: a dual role for CRP in the activation of an Escherichia coli promoter depending on MalT and CRP.

Activation of the Escherichia coli malEp promoter relies on the formation of a higher order structure involving cooperative binding of MalT to promoter-proximal and promoter-distal sites as well as CRP binding to three sites located in between. MalT is the primary activator and one function of CRP is to facilitate cooperative binding of MalT to its cognate sites by bending the intervening DNA. It is shown here that CRP also participates directly in malEp activation. This function is carried out by the molecule of CRP bound to the CRP site centered at -139.5 (CRP site 3). This molecule of CRP recruits RNA polymerase by promoting the binding of the RNA polymerase alpha subunit C-terminal domain (alphaCTD) to DNA immediately downstream from CRP site 3, via a contact between alphaCTD and activating region I of CRP. The action of MalT and CRP at malEp hence provides the example of a novel and complex mechanism for transcriptional synergy in prokaryotes whereby one activator both helps the primary activator to form a productive complex with promoter DNA and interacts directly with RNA polymerase holoenzyme.

ATP-Binding Cassette Transporters↗

PubMed2000

A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, self-associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer. In vivo studies have revealed that MalT action is negatively controlled by the MalY protein. Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity. We show that MalY inhibits transcription activation by MalT in a purified transcription system. In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild-type MalT to repression by MalY, as observed in vivo. We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT. Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding.

Bacterial Proteins↗

PubMed1999

Self-association of the Escherichia coli transcription activator MalT in the presence of maltotriose and ATP.

MalT, the transcriptional activator of the Escherichia coli maltose regulon, binds the MalT-dependent promoters and activates transcription initiation only in the presence of maltotriose and ATP (or adenylyl imidodiphosphate (AMP-PNP)). Cooperative binding of MalT to the array of cognate sites present in the MalT-dependent promoters suggests that promoter binding involves MalT oligomerization. Gel filtration and sedimentation experiments were used to analyze the quaternary structure of MalT in solution in the absence or presence of maltotriose and/or AMP-PNP, ATP, or ADP. The protein is monomeric in the absence of ligands and in the presence of ADP. In the presence of maltotriose, AMP-PNP, or ATP only, the protein self-associates, but a large fraction of the protein remains monomeric. In the presence of both maltotriose and AMP-PNP (ATP or ADP), the protein is essentially oligomeric, with the difference being that the oligomerization is less favored in the presence of ADP + maltotriose than in the presence of AMP-PNP + maltotriose. We present evidence that the association pathway comprises the following steps: monomers --> dimers --> (MalT)(n) --> aggregates, where 3 </= n </= 6. From these data, we conclude that the role of maltotriose and ATP as positive effectors is to induce the multimerization of MalT, and hence its cooperative binding to the mal promoters.

Adenosine Diphosphate↗

PubMed1998

H-NS and StpA proteins stimulate expression of the maltose regulon in Escherichia coli.

The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in Escherichia coli. Its role in gene regulation is manifested by the increased expression of several gene products in hns mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with hns mutant strains and derivatives also deficient in the stpA gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and lamB mRNA were greatly reduced in hns and hns-stpA mutant strains. The same results were obtained when we monitored the amount of transcription from the malEFG operon. The lamB gene is situated in the malKlamBmalM operon, which forms a divergent operon complex together with the malEFG operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a malT-lacZ translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in hns and hns-stpA mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.

ATP-Binding Cassette Transporters↗

PubMed1996

On the role of the multiple regulatory elements involved in the activation of the Escherichia coli malEp promoter.

Activation of malEp and malKp, two divergent promoters from Escherichia coli, depends on the synergistic action of MalT and CRP. The reaction involves a common regulatory region located in between and comprising multiple binding elements for both regulatory proteins. The binding of MalT and CRP to this region is known to result in the formation of a higher-order structure that is responsible for malKp activation. This paper presents genetic data which together with previous results, provide compelling evidence that this higher-order structure is also responsible for malEp activation. The role(s) that this structure or elements thereof play in the activation of malEp is analysed by monitoring both the occupancy of the proximal MalT sites (sites 1 and 2) and the activity of different malEp variants in strains containing increasing amounts of active MalT. A truncated malEp promoter comprising only MalT sites 1 and 2 forms a minimal MalT-dependent promoter whose activity is limited by the occupancy of these sites. One role of the higher-order structure formed by MalT and CRP when bound to the entire regulatory region is to ensure high occupation of MalT sites 1 and 2, but it is not its only function. Some elements of this structure, namely the CRP site 1, located at -76.5, and the distal MalT sites, seem to play a direct role in malEp activation besides their participation in the assembly of the higher-order structure.

ATP-Binding Cassette Transporters↗

PubMed1994

CRP induces the repositioning of MalT at the Escherichia coli malKp promoter primarily through DNA bending.

Activation of transcription initiation at the Escherichia coli malKp promoter requires the repositioning of MalT, the primary activator, from a set of non-productive sites to a set of productive sites, which is staggered by 3 bp. Occupation of the latter relies on the formation of a higher order structure involving distal MalT sites and the binding of CRP (cAMP receptor protein) to three sites located in the intervening region. We show here that one can successfully replace all of the CRP sites by the binding site of another DNA-bending protein, integration host factor, or by a sequence-directed bend without altering the process of malKp activation. This observation indicates that CRP action at malKp does not involve critical interactions with MalT and that CRP promotes MalT repositioning primarily through DNA bending. This structural role of CRP differs markedly from its role in the activation of the lac promoter.

Bacterial Proteins↗

PubMed1993

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.

Bacterial Proteins↗

PubMed1991

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.

Bacterial Proteins↗

PubMed1991

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.

Bacterial Proteins↗

PubMed1991

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.

Bacterial Proteins↗

PubMed1989

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.

Bacterial Proteins↗

PubMed1989

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.

Adenosine Triphosphatases↗

PubMed1988

Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex.

During lambda integration, Int recombinase must specifically bind to and cut attachment sites on both the viral and host chromosomes. We show here by foot-printing and by a novel cleavage assay that the bacterial attachment site, attB, cannot stably bind Int in competition with other DNAs. Instead, during recombination reactions, attB obtains its Int by collision with the intasome, a nucleoprotein assembly that forms on the viral attachment site, attP. Our cleavage assay also shows that the capture of attB by the attP intasome does not depend on DNA homology between the two sites; synapsis is governed solely by protein-protein and protein-DNA interactions.

Attachment Sites, Microbiological↗

PubMed1988

[Comparison of the retention time of particles of dehydrated lucerne and of the liquid phase of digesta in the digestive tract of sheep].

The mean retention time (MRT) of particles of ground and pelleted dehydrated lucerne was compared to that of the liquid phase in different parts of the sheep digestive tract. It was not different in the reticulo-rumen but it was greater in the stomachs and the whole tract, mainly because of a faster rate of passage of fluid in the abomasum and small intestine.

Animals↗

PubMed1987

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.

Adenosine Triphosphate↗

PubMed1987

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.

Enzyme Induction↗

PubMed1986

The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination.

Lambda integrative recombination depends on supercoiling of the phage attachment site, attP. Using dimethylsulfate protection and indirect end-labeling, the interaction of the recombination proteins Int and IHF with supercoiled and linear attP has been studied. Supercoiling enhances the binding of Int to attP, but not if a truncated attP site is employed or if IHF is omitted. We reason that the altered affinity reflects the formation of a higher-order nucleoprotein structure, an "attP intasome," that involves Int and IHF assembly of both arms of attP into a wrapped configuration. The good correlation between the degree and sign of supercoiling needed to promote recombination and that needed for the "attP intasome" indicates that the primary role of supercoiling is to drive the formation of the wrapped structure.

Bacterial Proteins↗

PubMed1985

Plasma catecholamine concentrations in lambs and calves during the perinatal period.

Concentrations of the plasma catecholamines (CA), adrenaline, noradrenaline and dopamine, were measured using a specific and sensitive radioenzymatic assay in 7 ewes and their single lambs chronically catheterized in utero between days 127 and 145 of gestation and during the first week of postnatal life. These concentrations were also measured during the first month after birth in 18 Holstein x Friesian calves, of which 9 were born spontaneously at term (278 days) and 9 by caesarean section on day 260 of gestation. Plasma CA concentrations in ewes showed no significant variations during the sampling period. Plasma adrenaline, noradrenaline and dopamine concentrations in lambs at birth rose significantly to 1 800 +/- 520, 3 782 +/- 781 and 575 +/- 90 pg/ml, respectively. These values remained higher than those measured in ewes until 2, 24 and 48 h after birth, respectively. On days 5 and 10 after delivery, plasma dopamine and noradrenaline concentrations were higher in calves delivered by caesarean section than in those born spontaneously at term, while plasma adrenaline concentration was never different between the two groups. These results indicate that the surge in plasma CA concentration normally occurring at birth might be delayed post partum in animals delivered before term. This might partly explain why these animals were less resistant to stress during the first days of postnatal life.

Aging↗