tailieunhanh - Báo cáo khoa học: Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro

The ability ofEntamoeba histolyticaTATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. . | iFEBS Journal Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro Guadalupe de Dios-Bravo1 2 Juan Pedro Luna-Arias3 Ana Maria Riveron4 Jose J Olivares-Trejo5 Cesar Lopez-Camarillo2 and Esther Orozco5 1 Programa de Biomedicina Molecular Escuela Nacionalde Medicina y Homeopatia del Institute Politecnico Nacional Mexico 2 Programa de Ciencias Genomicas Universidad de la Ciudad de Mexico Mexico 3 Departamento de Biologia Celular Centro de Investigacion y de Estudios Avanzados Mexico 4 Departamento de Biologia Molecular Centro Nacionalde Investigacion Cientifica CNIC Habana Cuba 5 Departamento de Patologia Experimental Centro de Investigacion y de Estudios Avanzados Mexico Keywords Entamoeba histolytica KD promiscuous DNA-binding activity TATA-binding protein TATA variants Correspondence Esther Orozco Departamento de Patologia Experimental Centro de Investigacion y de Estudios Avanzados IPN. C. P. 07360 Mexico D. F. Fax 52 55 57477108 Tel 52 55 50613800 ext 5642 E-mail esther@ Received 23 June 2004 revised 8 December 2004 accepted 11 January 2005 doi The ability of Entamoeba histolytica TATA binding protein EhTBP to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts NE and the TATTTAAA oligonucleotide which corresponds to the canonical TATA box for E. histolytica was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants .