## 1 Introduction  
Since the discovery of cAMP by [PERSON] and [PERSON] (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 (also 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 uM in the presence of glycerol, and 0.4 and 1.5 uM in the presence of glucose [6, 7]. The binding of cAMP to CRP shows negative cooperativity under a low ionic strength, although this is still debated. The dissociation constants for the first cAMP binding to CRP have been reported as 25 and 11 uM by equilibrium dialysis using radioactive cAMP [8, 9]. We describe a simple method to determine the binding affinity of ligands to proteins. Since the binding of cAMP to CRP has been studied extensively, previous studies using radioactive ligands may serve as a reference to corroborate the method described in this study.  
At least 12 nucleoid-associated proteins, which play a role in DNA packaging such as eukaryotic histone proteins have been described in _Escherichia coli_[10]. Among them, histone-like nucleoid structuring protein (H-NS) is involved in the biogenesis of flagella; H-NS mutation reduces the transcription of flhD and fliA genes, and thereby results in the complete loss of flagella [11]. The expression of approximately 5% of the genes is altered in the H-NS mutant strain. In most cases, H-NS appears to repress gene expression [12]. Transcription of the H-NS gene is down-regulated by ppGpp, the signaling molecule in bacterial stringent responses [13]. H-NS is an abundant protein, about 20,000 copies of which are present in each cell [14; 15]. The binding effect of abundant proteins to signaling molecules may have a more significant impact in cells by sequestering a large portion of signal molecules.  
The stringent response is mediated by guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (ppGpp) in response to nutrient (phosphate, iron, carbon, and fatty acid) deprivation [16; 17]. (p)ppGpp also serves as a global regulator in replication, transcription, and translation, including the expression of about 1400 genes [18; 19; 20; 21]. Despite its importance in bacterial physiology and gene regulation and nearly half a century of study, the mechanisms concerning how ppGpp acts on stringent responses remains incompletely understood since the full set of effector proteins is unknown. Recently, CRP has been identified as a ppGpp binding protein in a study utilizing a photo-crosslinking ppGpp analog [22]. H-NS is also captured to a ppGpp-immobilized resin (unpublished data). Nevertheless, the binding of ppGpp to CRP and H-NS has yet to be reported.  
In this study, ppGpp binding to CRP and H-NS was determined by fluorescence spectroscopy, while cAMP-binding to CRP was used as a reference. The dissociation constant of cAMP to CRP was similar to those reported previously, implying that the method described here is valid. 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_.