Curation Information

Publication: Illumination stimulates cAMP receptor protein-dependent transcriptional activation from regulatory regions containing class I and class II promoter elements in Synechocystis sp. PCC 6803.;Hedger J, Holmquist PC, Leigh KA, Saraff K, Pomykal C, Summers ML;Microbiology (Reading, England) 2009 Sep; 155(Pt 9):2994-3004 [19542007]
TF: CRP [P74171, view regulon]
Reported TF sp.: Synechocystis sp. PCC 6803
Reported site sp.: Synechocystis sp. PCC 6803
Created by: Matthew Coveyou
Curation notes: -

Experimental Process

Previous studies identified 11 potential Crp-dependent genes and their binding sites in Synechocystis sp. PCC 6803. EMSA of oligonucleotide fragments containing these sites demonstrated specific binding in three: slr1351 (murF), slr1874 (AT103), and slr0442 (hypothetical protein). A Crp- mutant was developed and tested to ensure expression was not affected by polar effects or non-target recombination. The mutant was then used in several different qRT-PCR assays, including variable illumination time, cAMP levels, and Crp expression. murF and AT103 expression was four and ten times higher (respectively) in the wild-type over the mutant after 1 hour of illumination with cAMP increase. Wild-type expression of slr0442 did not appear to increase over illumination time, but further investigation suggested increased expression was negated by increased simultaneous mRNA degradation; consequently, wild-type expression of slr0442 was five times greater than mutant expression. Results confirmed Crp-dependent activation of all three genes. GFP reporter assays in wild-type and Crp-mutant E. coli paralleled results in Synechocystis, except in the case of AT103, whose promoter did not drive transcription in E. coli. RACE determined transcription start sites for murF, AT103, and slr0442, and multiple sequence alignment of the murF promoter with homologous proteins from Crocosphaera and Cyanothece species identified a conserved region in agreement with that determined for AT103 and slr0442.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

GGTGATCTAGATCACA
TGTGAGAATAATCACA
TGTGATCCAGATCACA

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence	Regulated genes	Experimental techniques	TF function	TF type
GGTGATCTAGATCACA	murF,	Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details GFP Promoter Fusion (ECO:0005636) GFP Promoter Fusion - ECO:0005636 Target promoter is inserted in front of a GFP gene, and the target TF is induced. Expression levels of GFP should thereby depend on the target TF. - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) Multiple sequence alignment (MSA) - ECO:0005556 MSA can be used to align promoter regions and highlight regions of conservation that may be indicative of transcription factor binding sites. - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	activator	not specified
TGTGAGAATAATCACA	AT103,	Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	activator	not specified
TGTGATCCAGATCACA	slr0442,	Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details GFP Promoter Fusion (ECO:0005636) GFP Promoter Fusion - ECO:0005636 Target promoter is inserted in front of a GFP gene, and the target TF is induced. Expression levels of GFP should thereby depend on the target TF. - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	activator	not specified