Biomedical subjects

L Søgaard-Andersen

Publications and source records attributed to L Søgaard-Andersen.

27 records · Page 2Linked to original sources

PubMed1991

Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli.

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode tandem binding sites for cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively low affinity; in the presence of cAMP-CRP, however, the repressor and activator bind cooperatively to the DNA. Here we have investigated this cooperativity by isolating mutants of the CRP protein that abolish CytR regulation without exhibiting a concomitant loss in their ability to activate transcription. Four different, single amino acid substitutions in CRP give rise to this phenotype. These amino acids lie in close proximity on the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of one of the CRP mutants show that it interacts with the DNA in a manner indistinguishable from wild-type CRP, whereas its interaction with CytR is perturbed. These results strongly indicate that cooperative DNA binding of CytR and cAMP-CRP is achieved through protein-protein interactions.

Bacterial Proteins↗

PubMed1991

Restored DNA-binding of the cAMP-CRP activator complex reestablishes negative regulation by the CytR repressor in the deoP2 promoter in Escherichia coli.

We have investigated in vivo the coupling between CytR regulation of the deoP2 promoter in Escherichia coli and the DNA-binding specificity of the cAMP-CRP (cAMP receptor protein) complex in order to obtain a more detailed picture of the role played by cAMP-CRP in CytR regulation. By introducing CRP proteins that exhibit an altered DNA binding specificity into a strain containing a mutant deoP2 promoter in which cAMP-CRP activation was decreased and CytR regulation completely abolished, we show that CytR regulation of this promoter can be reestablished by restored the DNA binding of the cAMP-CRP complex. Hence, CytR regulation of deoP2 can be modulated by simply varying DNA binding of cAMP-CRP. These data confirm the crucial role played by the cAMP-CRP activator complex in CytR regulation of the deoP2 promoter.

Bacterial Proteins↗

PubMed1991

A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon.

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.

Bacterial Proteins↗

PubMed1991

Design of cAMP-CRP-activated promoters in Escherichia coli.

We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by cAMP-CRP. Based on these observations, we propose that cAMP-CRP-activated promoters can be created by correctly aligning a CRP target and a -10 hexamer. This idea has been successfully tested by converting both a CRP-independent promoter and a sequence resembling the consensus -10 hexamer to strongly cAMP-CRP-activated promoters.

Base Sequence↗

PubMed1991

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12.

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.

Amino Acid Sequence↗

PubMed1990

The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12.

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of CytR are discussed in light of these findings.

Base Sequence↗

PubMed1990

Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli.

We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the -35 region, and is sufficient for activation; the second site, CRP-2, centred around -93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP-CRP complexes.

Bacterial Proteins↗

PubMed1985

Two functions of the E protein are key elements in the plasmid F replication control system.

By using a plasmid carrying a translational fusion between the E gene of the IncFI plasmid F and the lacZ gene, we located the operator of the autogenously regulated E gene to an inverted repeat overlapping the E-gene promoter and showing perfect homology to part of the sequence found in all the direct repeats of two regions exerting an inhibitory effect on F replication, incB and incC. Excess E protein provided in trans to an F plasmid increased the replication frequency of the F plasmid. This stimulatory effect was counteracted by increased dosages of incB or incC. A model is proposed for the replication control system of F in which the key elements are autoregulation of E-gene expression and titration of E protein by incB and incC.

Bacterial Proteins↗

PubMed1984

Regulated expression of a gene important for replication of plasmid F in E. coli.

Fusions between the gene encoding the E protein of the IncFI plasmid F and the lac genes were constructed. Analysis of the expression of beta-galactosidase from these fusions shows that the promoter for the E protein gene is located between the incB region and the structural gene for the E protein. Near this promoter is a regulatory site on which a negative control element acts. Most likely the E protein itself acts as a repressor of E gene expression and thus autoregulates its own expression. No other gene products seem to affect the expression of the E protein gene.

Bacterial Proteins↗