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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.
Mol Microbiol 1998 Sep
PMID:A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli. 978 86

A Bayesian procedure for the simultaneous alignment and classification of sequences into subclasses is described. This Gibbs sampling algorithm iterates between an alignment step and a classification step. It employs Bayesian inference for the identification of the number of conserved columns, the number of motifs in each class, their size, and the size of the classes. Using Bayesian prediction, inter-class differences in all these variables are brought to bare on the classification. Application to a superfamily of cyclic nucleotide-binding proteins identifies both similarities and differences in the sequence characteristics of the five subclasses identified by the procedure: 1) cNMP-dependent kinases, 2) prokaryotic cAMP-dependent regulatory proteins, CRP-type, 3) prokaryotic regulatory proteins, FNR-type, 4) cAMP gated ion channel proteins of animals, and 5) cAMP gated ion channels of plants.
Proc Int Conf Intell Syst Mol Biol 1998
PMID:Bayesian protein family classifier. 978 18

The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.
J Mol Biol 1998 Dec 18
PMID:Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase sigma70 subunit. 987 55

Erwinia chrysanthemi 3937 synthesizes an exopolysaccharide (EPS) composed of rhamnose, galactose, and galacturonic acid. Fourteen transcriptional fusions in genes required for EPS synthesis, named eps, were obtained by Tn5-B21 mutagenesis. Eleven of them are clustered on the chromosome and are repressed by PecT, a regulator of pectate lyase synthesis. In addition, expression of these fusions is repressed by the catabolite regulatory protein, CRP, and induced in low osmolarity medium. The three other mutations are located in genes that are not regulated by pecT. A 13-kb DNA fragment containing pecT-regulated eps genes has been cloned. All the genes identified on this fragment are transcribed in the same orientation and could form a large operon. The promoter region of this operon has been sequenced. It contains a JUMP-start sequence, a sequence required for the expression of polysaccharide-associated operons. E. chrysanthemi 3937 produces a systemic soft rot on its host Saintpaulia ionantha. An eps mutant was less efficient than the wild-type strain in initiating a maceration symptom, suggesting that production of EPS is required for the full expression of the E. chrysanthemi virulence.
Mol Plant Microbe Interact 1999 Jan
PMID:The PecT repressor coregulates synthesis of exopolysaccharides and virulence factors in Erwinia chrysanthemi. 988 92

Global regulatory circuits of the type mediated by CRP and FNR in Escherichia coli were sought in Lactococcus lactis to provide a basis for redirecting carbon metabolism to specific fermentation products. Using a polymerase chain reaction (PCR) approach, two genes (flpA and flpB) encoding FNR-like proteins (FlpA and FlpB) with the potential for mediating a dithiol-disulphide-dependent regulatory switch, were identified. Transcript analysis indicated that they are distal genes of two paralogous operons, orfX-orfY-flp, in which the orfX and orfY genes were predicted to encode binding domain components of cation ATPases and storage proteins respectively. The corresponding promoters were each associated with a potential FNR site (TTGAT----ATCAA) at positions +4.5 (flpA operon) and -42.5 (flpB operon), suggesting that the respective operons might be negatively and positively autoregulated. The incomplete open reading frames (orfWA/B) located upstream of each operon were predicted to encode additional components of paralogous cation ATPases. No phenotypic effects were detected in flpA and flpB single mutants, but the double mutant had a lower intracellular zinc content, an increased sensitivity to hydrogen peroxide and an altered polypeptide profile (as determined by two-dimensional gel electrophoresis): formate production was not affected. It was concluded tentatively that FlpA and FlpB regulate overlapping modulons, including systems concerned with zinc uptake, in response to metal ion or oxidative stress.
Mol Microbiol 1999 Mar
PMID:Two operons that encode FNR-like proteins in Lactococcus lactis. 1020 Sep 70

The dadAX operon is expressed by multiple promoters that are repressed by leucine-responsive regulatory protein (Lrp) and activated by cyclic AMP-CRP. In previous work, we found that alanine or leucine acted as inducers to antagonize Lrp repression of the three major promoters directly. Here, we identify 11 Lrp binding sites located within 350 bp of dad DNA. A mutational analysis, coupled with in vivo and in vitro transcription experiments, indicated that Lrp sites that overlap the dad promoters were involved in repression. In contrast, sites upstream of the promoters did not appear to be necessary for repression, but were required for activation by Lrp plus alanine or leucine of one of the major dad promoters, P2. This activation by alanine or leucine was not simply relief of repression, as P2 transcription from a constitutive template was increased fivefold compared with the basal level of transcription found in the absence of Lrp and the co-activator cyclic AMP-CRP. Alanine or leucine decreased the affinity of Lrp to repressor sites, while having little or no effect on the binding of Lrp to activator sites. This differential effect of alanine and leucine on Lrp binding helps to explain how these modifiers influence both repression and activation of the dad operon.
Mol Microbiol 1999 Apr
PMID:Lrp binds to two regions in the dadAX promoter region of Escherichia coli to repress and activate transcription directly. 1021 57

Previously, we have cloned and characterized the pir (plant inducible regulator) gene, which is responsible for hyperinduction of the synthesis of an isozyme of pectate lyase (PLe) in Erwinia chrysanthemi EC16 in the presence of potato extract and sodium polypectate (NaPP). The Pir protein purified from Escherichia coli overexpressing pir is able to bind to the promoter region of pir as a dimer. Self-regulation of pir by its own translational product (Pir) was suggested from the findings that Pir binds at the promoter region of pir and that the hyperinduction of the pirlux construct in response to plant extract was observed only in pir+ but not in pir mutant EC16. Thus, hyperinduction of PLe was thought to be mainly due to overproduction of Pir. On the other hand, KdgR and PecS, which have been reported to be the major regulatory proteins for the synthesis of pectic enzymes, did not bind to the promoter region of pir. Thus, the regulation of Pir synthesis seems to be independent of KdgR and PecS. Also, its expression was insensitive to catabolite repression as predicted from failure of cyclic AMP (cAMP)-CRP (cAMP recognizing protein) to bind at the pir promoter region.
Mol Plant Microbe Interact 1999 May
PMID:Self-regulation of pir, a regulatory protein responsible for hyperinduction of pectate lyase in Erwinia chrysanthemi EC16. 1022 71

Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules. The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators. Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytRSt) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytREc) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytRSt to CytREc, revealing that serine substitutions of only two residues (G131 and A152) in CytREc is required to make CytREc sensitive to uridine. In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytRSt that appear to be important for the response to uridine. The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes.
J Mol Biol 1999 Apr 23
PMID:Protein-ligand interaction: grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR. 1032 34

The LIM domain is a conserved cysteine and histidine-containing structural module of two tandemly arranged zinc fingers. It has been identified in single or multiple copies in a variety of regulatory proteins, either in combination with defined functional domains, like homeodomains, or alone, like in the CRP family of LIM proteins. Structural studies of CRP proteins have allowed a detailed evaluation of interactions in LIM-domains at the molecular level. The packing interactions in the hydrophobic core have been identified as a significant contribution to the LIM domain fold, whereas hydrogen bonding within each single zinc binding site stabilizes zinc finger geometry in a so-called "outer" or "indirect" coordination sphere. Here we report the solution structure of a point-mutant of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein CRP2, CRP2(LIM2)R122A, and discuss the structural consequences of the disruption of the hydrogen bond formed between the guanidinium side-chain of Arg122 and the zinc-coordinating cysteine thiolate group in the CCHC rubredoxin-knuckle. The structural analysis revealed that the three-dimensional structure of the CCHC zinc binding site in CRP2(LIM2)R122A is adapted as a consequence of the modified hydrogen bonding pattern. Additionally, as a result of the conformational rearrangement of the zinc binding site, the packing interactions in the hydrophobic core region are altered, leading to a change in the relative orientation of the two zinc fingers with a concomitant change in the solvent accessibilities of hydrophobic residues located at the interface of the two modules. The backbone dynamics of residues located in the folded part of CRP2(LIM2)R122A have been characterized by proton-detected(15)N NMR spectroscopy. Analysis of the R2/R1ratios revealed a rotational correlation time of approximately 6.2 ns and tumbling with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.43). The relaxation data were also analyzed using a reduced spectral density mapping approach. As in wild-type CRP2(LIM2), significant mobility on a picosecond/nanosecond time-scale was detected, and conformational exchange on a microsecond time-scale was identified for residues located in loop regions between secondary structure elements. In summary, the relative orientation of the two zinc binding sites and the accessibility of hydrophobic residues is not only determined by hydrophobic interactions, but can also be modified by the formation and/or breakage of hydrogen bonds. This may be important for the molecular interactions of an adaptor-type LIM domain protein in macromolecular complexes, particularly for the modulation of protein-protein interactions.
J Mol Biol 1999 Oct 01
PMID:Mutational analysis and NMR spectroscopy of quail cysteine and glycine-rich protein CRP2 reveal an intrinsic segmental flexibility of LIM domains. 1052 13

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti-repressor against KdgR.
Mol Microbiol 1999 Nov
PMID:Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator. 1056 5


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