Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by dynamic light scattering and time-resolved fluorescence anisotropy methods.
Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.
Błaszczyk
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Department of Physical Biochemistry, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Polit
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Guz
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Wasylewski
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D002918:Chymotrypsin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D008027:Light; D011401:Promoter Regions, Genetic; D012542:Scattering, Radiation; D013997:Time Factors
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D000242:Cyclic AMP; D002918:Chymotrypsin