Fluorescence Quenching Studies of Conformational Changes Induced by cAMP and DNA Binding to Heterodimer of Cyclic AMP Receptor Protein from Escherichia coli

Abstract

In Escherichia coli, cyclic AMP receptor protein (CRP) is known to regulate the transcription of about 100 genes. The signal to activate CRP is the binding of cyclic AMP. In this study the fluorescence quenching measurements were used to observe conformational changes in the structure of CRP after binding of cAMP and DNA. We used the constructed CRP heterodimer, which contains only a single Trp13 residue localized in the N-terminal domain of one CRP subunit. We propose that apo-CRP subunits exist in a solution in one conformational state and it changes after the ligand binding. We also suggest that the signal transmission upon binding of cAMP is possible not only from the N-terminal domain to C-terminal domain but also in the opposite direction after binding of specific DNA sequence, both with and without cAMP. Thereby it can influence on the CRP's interaction with RNA polymerase and the genes expression.

In vitro fluorescence studies of transcription factor IIB-DNA interaction

“…Our findings, which are consistent with the results obtained for prokaryotic transcription factor cAMP receptor protein (Fic et al, 2007), may lead to a more general conclusion. In the transcription initiation process, subtle changes in the structure and dynamics of the various transcription factors, depending on reciprocal interactions, play an important role, apart from the obvious structural aspects of the interaction between transcription factors and the RNAP, leading to suitable geometry of the complex formation, mutual affinity of the individual components or the kinetics of their interactions.…”

cAMP Receptor Protein from <i>Escherichia coli</i> as a Model of Signal Transduction in Proteins – A Review

“…Binding of DNA promoter sequences to the C-terminal domain causes changes in the overall protein structure, both in the presence and in the absence of the ligand. It can be concluded that signal transmission from the C-terminal domain to the N-terminal domain may play a crucial role in gene expression [Fic et al, 2007]. Moreover, CRP binding to the specific DNA sequences depends not only on the presence of the consensus sequence but also on the presence of proper flanking sequences of DNA [Dai et al, 2004].…”

Site‐directed fluorescence labeling of intrinsically disordered region of human transcription factor YY1: The inhibitory effect of zinc ions

“…The presence of regions that can modify the labeling kinetics, that is, rich in histidine or acidic residues—capable of coordinating metal ions, may be used to provide selective or double labeling. Labeling of the disordered regions, which are highly flexible and dynamic, provides the possibility for unhindered rotation of the label, which makes it especially attractive in a number of applications, for example for FRET measurements, where it provides appropriate κ value, allowing for direct measurement of distances.…”

“…In the latter case, the location of the fluorescence dye is crucial, since the monitored parameters of the structure are usually limited to the microenvironment of the fluorophore. Among others, it is possible to determine the exposure of the fluorophore by direct measurement of the fluorescence emission spectrum of dyes with solvatochromic properties, or by using a fluorescence quenching for fluorophores that do not necessarily have this property . It is possible to determine the rotational dynamics of a macromolecule or its fragments by measuring stationary or time‐resolved fluorescence anisotropy .…”