Dissecting protein:protein interactions between transcription factors with an RNA aptamer.
Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016356:Gene Products, tax / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000037:antagonists & inhibitors / Q000378:metabolism*; D026362:Serum Response Factor; D014157:Transcription Factors / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D016356:Gene Products, tax; D016328:NF-kappa B; D009687:Nuclear Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D012313:RNA