Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro.
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 found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (+/-0.39) x 10(-11) and 1.60 (+/-0.37) x 10(-10) m. TATTTAAA and TAT_ _AAA motifs presented the lowest KD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding.
de Dios-Bravo
Guadalupe
G
Programa de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía del Instituto Politécnico Nacional, Mexico.
Luna-Arias
Juan Pedro
JP
Riverón
Ana María
AM
Olivares-Trejo
José J
JJ
López-Camarillo
César
C
Orozco
Esther
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004748:Entamoeba histolytica / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016385:TATA Box / Q000235:genetics / Q000502:physiology*; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015800:Protozoan Proteins; D035181:TATA-Box Binding Protein