Biomedical subjects

S Garges

Publications and source records attributed to S Garges.

10 recordsLinked to original sources

PubMed1992

Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.

Allosteric Regulation↗

PubMed1992

Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.

Chloramphenicol O-Acetyltransferase↗

PubMed1991

A mutation defining ultrainduction of the Escherichia coli gal operon.

Tn10 insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a delta galR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.

DNA Transposable Elements↗

PubMed1991

Further inducibility of a constitutive system: ultrainduction of the gal operon.

In wild-type Escherichia coli, expression of the gal operon is negatively regulated by the Gal repressor and is induced 10- to 15-fold when the repressor is inactivated by an inducer. In strains completely deleted for galR, the gene which encodes the Gal repressor, the operon is derepressed by only 10-fold without an inducer. But this derepression is increased further by threefold during cell growth in the presence of an inducer, D-galactose or D-fucose. This phenomenon of extreme induction in the absence of Gal repressor is termed ultrainduction--a manifestation of further inducibility in a constitutive setup. Construction and characterization of gene and operon fusion strains between galE and lacZ, encoding beta-galactosidase as a reporter gene, show that ultrainduction occurs at the level of transcription and not translation. Transcription of the operon, from both the cyclic AMP-dependent P1 and the cyclic nucleotide-independent P2 promoters, is subject to ultrainduction. The wild-type galR+ gene has an epistatic effect on ultrainducibility: ultrainduction is observed only in cells devoid of Gal repressor protein. Titration experiments show the existence of an ultrainducibility factor that acts like a repressor and functions by binding to DNA segments (operators) to which Gal repressor also binds to repress the operon.

DNA Mutational Analysis↗

PubMed1990

Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E. coli cAMP receptor protein.

Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP. In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP. To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined. The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x 10(4) M-1; CRP*598, 3.8 x 10(5) M-1. The K values obtained for cGMP binding are: CRP, 2.9 x 10(4) M-1; CRP*598, 2.7 x 10(4) M-1. The results indicate that the affinity of CRP and CRP*598 for cGMP is relatively unchanged while the affinity of CRP*598 for cAMP is approximately twenty times greater than that shown by CRP. Binding of cAMP by CRP and cGMP by CRP or CRP*598 exhibits slight negative cooperativity. The major difference seen is that CRP*598 binds cAMP with strong positive cooperativity. The importance of the unsubstituted N6 position of the adenine moiety is also shown by the similar affinity of both forms of CRP for N6-butyryl cAMP. The cAMP binding properties evinced by CRP*598 suggest that its intrinsically altered conformation may be related to that assumed by CRP in a CRP-DNA or a cAMP-CRP-DNA complex.

Adenosine↗

PubMed1990

Positive control.

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Allosteric Regulation↗

PubMed1988

Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase.

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [32P]lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding. In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.

Cyclic AMP↗

PubMed1988

Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations.

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.

Allosteric Site↗

PubMed1985

Sites of allosteric shift in the structure of the cyclic AMP receptor protein.

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.

Amino Acid Sequence↗

PubMed1979

Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.

Aerobiosis↗