Abstract
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.
MeSH Terms
Base Sequence
Chromosome Deletion
Cyclic AMP/metabolism
DNA, Bacterial/genetics,isolation & purification
Escherichia coli/genetics
Escherichia coli Proteins
Molecular Sequence Data
Mutation
Oligonucleotide Probes
Plasmids
Promoter Regions, Genetic
Receptors, Cyclic AMP/metabolism
Recombinant Fusion Proteins/metabolism
Repressor Proteins/genetics,metabolism
Restriction Mapping
beta-Galactosidase/genetics,metabolism
Chemicals
CytR protein, E coli
DNA, Bacterial
Escherichia coli Proteins
Oligonucleotide Probes
Receptors, Cyclic AMP
Recombinant Fusion Proteins
Repressor Proteins
Cyclic AMP
beta-Galactosidase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Søgaard-Andersen L
Department of Molecular Biology, Odense University, Denmark.
Martinussen J
Møllegaard N E
Douthwaite S R
Valentin-Hansen P
References (27)
27 references, click to expand
-
[Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12].
Genetika. 1989 Mar;25(3):438-47
PMID: 2668121
-
Improved plasmid vectors for the isolation of translational lac gene fusions.
Gene. 1984 Nov;31(1-3):269-73
PMID: 6098531
-
Mutations in the Escherichia coli operon that define two promoters and the binding site of the cyclic AMP receptor protein.
J Mol Biol. 1982 Jan 15;154(2):211-27
PMID: 6804633
-
Cyclic AMP receptor protein: role in transcription activation.
Science. 1984 May 25;224(4651):831-8
PMID: 6372090
-
CRP/cAMP- and CytR-regulated promoters in Escherichia coli K12: the cdd promoter.
Mol Microbiol. 1989 Oct;3(10):1385-90
PMID: 2575702
-
Plasmid vehicles for direct cloning of Escherichia coli promoters.
J Bacteriol. 1979 Nov;140(2):400-7
PMID: 387734
-
DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor.
EMBO J. 1986 Aug;5(8):2015-21
PMID: 3019678
-
Nucleotide sequence of the CytR regulatory gene of E. coli K-12.
Nucleic Acids Res. 1986 Mar 11;14(5):2215-28
PMID: 3515317
-
Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli.
EMBO J. 1985 Dec 1;4(12):3333-8
PMID: 3004952
-
The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.
Nucleic Acids Res. 1985 Dec 20;13(24):8765-85
PMID: 3001650
-
Structure and function of the intercistronic regulatory deoC-deoA element of Escherichia coli K-12.
EMBO J. 1984 Jan;3(1):179-83
PMID: 6323164
-
Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter.
Gene. 1984 Apr;28(1):45-54
PMID: 6329915
-
Transcriptional repression of eukaryotic promoters.
Cell. 1989 Nov 3;59(3):405-8
PMID: 2572326
-
Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator.
J Biol Chem. 1988 Dec 25;263(36):19653-61
PMID: 3058704
-
Protein-DNA recognition.
Annu Rev Biochem. 1984;53:293-321
PMID: 6236744
-
Purification and characterization of the deoR repressor of Escherichia coli.
EMBO J. 1989 Jan;8(1):325-31
PMID: 2653814
-
The structure of tandem regulatory regions in the deo operon of Escherichia coli K12.
EMBO J. 1982;1(3):317-22
PMID: 16453417
-
DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.
Nucleic Acids Res. 1978 Sep;5(9):3157-70
PMID: 212715
-
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene. 1985;33(1):103-19
PMID: 2985470
-
Tandem CRP binding sites in the deo operon of Escherichia coli K-12.
EMBO J. 1982;1(9):1049-54
PMID: 6329724
-
The tac promoter: a functional hybrid derived from the trp and lac promoters.
Proc Natl Acad Sci U S A. 1983 Jan;80(1):21-5
PMID: 6337371
-
MalI, a novel protein involved in regulation of the maltose system of Escherichia coli, is highly homologous to the repressor proteins GalR, CytR, and LacI.
J Bacteriol. 1989 Sep;171(9):4888-99
PMID: 2670898
-
Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides.
Biochemistry. 1988 Jul 12;27(14):5257-61
PMID: 2844254
-
Sequencing end-labeled DNA with base-specific chemical cleavages.
Methods Enzymol. 1980;65(1):499-560
PMID: 6246368
-
Long-range cooperativity between gene regulatory sequences in a prokaryote.
Nature. 1987 Feb 26-Mar 4;325(6107):823-6
PMID: 3547140
-
Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli.
J Bacteriol. 1951 Sep;62(3):293-300
PMID: 14888646
-
Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.
Mol Gen Genet. 1978 Feb 16;159(2):191-202
PMID: 204861