DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy.
Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA.
Sevenich
F W
FW
Deutsches Krebsforschungszentrum, Abteilung Biophysik der Makromolekule, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Langowski
J
J
Weiss
V
V
Rippe
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D051922:PII Nitrogen Regulatory Proteins; D011485:Protein Binding; D011487:Protein Conformation; D012475:Salmonella / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli