Abstract
The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.
Highlights
Since the discovery of cAMP by Sutherland and Rall (1958), molecules in combination with cAMP receptor protein (CRP) and adenylate cyclase have served as the model for signal transduction pathways [1]
PpGpp binding to CRP and histone-like nucleoid structuring protein (H-NS) was determined by fluorescence spectroscopy, while cAMP-binding to CRP was used as a reference
We report here for the first time that CRP and H-NS bind to ppGpp with ‘high’ affinity, suggesting that their binding may play a role in the regulation of gene expression in Escherichia coli
Summary
Since the discovery of cAMP by Sutherland and Rall (1958), molecules in combination with cAMP receptor protein (CRP) and adenylate cyclase have served as the model for signal transduction pathways [1]. A CRP dimer binds two cAMPs with an anti-conformation and two additional cAMPs with a syn-conformation (with a substantially lower affinity). The anti-cAMP binding is responsible for the global allosteric transition of CRP [2,3]. In. Escherichia coli, CRP ( referred to as catabolite gene activator protein, CAP) regulates the transcription of about 400 genes [4,5]. The cellular concentrations of cAMP in an Escherichia coli strain have been reported to vary between 1.5 and 5 μM in the presence of glycerol, and 0.4 and 1.5 μM in the presence of glucose [6,7]. The binding of cAMP to CRP shows negative cooperativity under a low ionic strength, this is still debated. The dissociation constants for the first cAMP binding to CRP have been reported as 25 and
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