Real-Time Interaction between ТВР and the TATA Box of the Human Triosephosphate Isomerase Gene Promoter in the Norm and Pathology

abS tract The TATA-binding protein (ТВР) is a key part of the transcription complex of RNA polymerase II. Alone or as a part of the basal transcription factor TFIID, TBP binds the TATA box located in the core region of the TATA-containing promoters of class II genes. Previously, we studied the effects of single nucleotide polymorphisms (SNPs) on ТВР/ТАТА-box interactions using gel retardation assay. It was demonstrated that most SNPs in the ТАТА boxes of some human gene promoters cause a 2- to 4-fold decrease in ТВР/ТАТА affinity, which is associated with an increased risk of hereditary diseases, such as β thalassemias of diverse severity, hemophilia B Leyden, myocardial infarction, thrombophlebitis, lung cancer, etc. In this work, the process of ТВР/ТАТА complex formation has been studied in real time by a stopped-flow technique using recombinant human TBP and duplexes, which were identical to the TATA box of the wild-type and a SNP-containing triosephosphate isomerase gene promoter and were fluorescently labeled by the Cy3/Cy5 FRET pair. It has been demonstrated for the first time that real-time binding of ТВР to the TATA box of the TPI gene promoter is complete within 10 s and is described by a single-stage kinetic model. The complex formation of ТВР with the wild-type TATA box occurs 5.5 times faster and the complex dissociation occurs 31 times slower compared with the SNPcontaining TATA box. Within the first seconds of the interaction, ТВР binds to and simultaneously bends the TATA box. Importantly, the TATA box of the wild-type TPI gene promoter requires lower TBP concentrations compared to the TATA box containing the -24Т → G SNP, which is associated with neurological and muscular disorders, cardiomyopathy, and other diseases.