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.
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Abbreviations
- cAMP:
-
3′,5′-cyclic adenosine monophosphate
- CRP:
-
cAMP receptor protein
- CRP W13–CRP:
-
CRP without tryptophan residues in one subunit and without Trp85 in the second subunit
- CRP S128A:
-
CRP mutant with serine residue in position 128 replaced with alanine in both subunits
- CRPW85A:
-
CRP mutant with tryptophan residue in position 85 replaced with alanine in both subunits
- AR:
-
activating region
- CD:
-
circular dichroism
- EDTA:
-
ethylenediaminetetraacetate
- FQRS:
-
fluorescence-quenching-resolved spectra
- gal :
-
a fragment of DNA sequence recognized by CRP in the galP1 promoter
- ICAP :
-
consensus DNA sequence recognized by CRP
- lac :
-
a fragment of DNA sequence recognized by CRP in the lacP1 promoter
- PAGE:
-
polyacrylamide gel electrophoresis
- RNAP:
-
polymerase RNA
- SDS:
-
sodium dodecyl sulfate
- HTH:
-
helix-turn-helix
- wt:
-
wild type
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Acknowledgments
We would like to thank Prof. Marta Dziedzicka-Wasylewska for the fruitful discussion and for all help. This work was supported by Grant 3PO4A 006 24 from the Ministry of Science and Informatics.
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Fic, E., Górecki, A. & Wasylewski, Z. Fluorescence Quenching Studies of Conformational Changes Induced by cAMP and DNA Binding to Heterodimer of Cyclic AMP Receptor Protein from Escherichia coli . Protein J 26, 457–466 (2007). https://doi.org/10.1007/s10930-007-9085-0
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DOI: https://doi.org/10.1007/s10930-007-9085-0