Detection of protein-DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) is a methodology to examine directly the translational diffusion of individual fluorescence-labeled molecules in solutions. Recent studies using FCS have quantified various bimolecular reactions without any need for amplification. To evaluate further the applicability of FCS, we studied the specific binding between proteins and DNA in crude biological samples. Using an automated FCS system that was recently developed in our laboratories and is capable of distinguishing two or more molecular species in a multicomponent analysis, we detected the binding of two representative transcription factors, activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), in nuclear extracts of HeLa cells quantitatively with each sequence-specific DNA. The binding rates of these specific interactions were markedly augmented when cells were treated with tumor necrosis factor alpha which is known to activate both AP-1 and NF-kappaB. We also observed the pyrrolidine-dithiocarbamate-induced reciprocal regulation of these transcription factors. These results indicated that FCS is a useful tool for the analysis of complex interactions of transcription factors with DNA even in crude cellular extracts, suggesting that it is a powerful methodology for the study of a wide variety of molecular events under various experimental conditions.
Kobayashi
Tamiyo
T
Life Science Group, Olympus Corp., Tokyo 192-8512, Japan. tami_kobayashi@ot.olympus.co.jp
Okamoto
Naoaki
N
Sawasaki
Tatsuya
T
Endo
Yaeta
Y
D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016328:NF-kappa B; D011506:Proteins; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D048031:JNK Mitogen-Activated Protein Kinases