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.
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||Gene diagram||Experimental techniques||TF function||TF type|