Multiple amino acid alignment of TATA-box binding protein 1 from <i>T</i>. <i>solium</i> (TsTBP1).

<p>TsTBP1 was aligned with <i>Sus scrofa</i> TBP1 (SsTBP1, GeneBank ID: XP_003361466.1), <i>Homo sapiens</i> TBP1 (HsTBP1, GeneBank ID: NP_003185.1), <i>E</i>. <i>granulosus</i> TBP1 (EgTBP1, GeneBank ID: CDS17003.1), <i>E</i>. <i>multilocularis</i> TBP1 (EmTBP1, GeneBank ID: CDJ04746.1). The NH₂-ter is enclosed in a box, and the remaining amino acid sequence corresponds to the COOH-terminal domain (COOH-ter). Identical amino acids are highlighted in gray background. Important residues that bind TATA-box are in red letters; transcription factor II A (TFIIA) in white; transcription factor II B (TFIIB) in blue; negative cofactor 2 (NC2) in orange and TBP1-associated factor 1 (TAF1) in yellow. Amino acid sequences used to produce the TsTBP probe and the synthetic peptides pTsTBP1-N and pTsTBP1-C are in small boxes and underlined, respectively. Letter X on <i>S</i>. <i>scrofa</i> TBP1 sequence means amino acids not identified. The symbols under the amino acids indicate: (-) absence and (:) homology of amino acids.</p

Electrophoretic mobility shift assay showing the interaction of wild type TsTBP1 pAT5 TATA-box probe.

<p>A) Lane 1: Labeled dsDNA-³²P probe without nuclear extract; lane 2: TsTBP1-pAT5 TATA-box interaction with <i>T</i>. <i>solium</i> nuclear extract; lanes 3, 4, and 5: competence with pAT5 TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 6: super-shift interaction using anti-pTsTBP1-N; lane 7: consensus TATA-box probe interaction with <i>T</i>. <i>solium</i> nuclear extract (used as positive control); lane 8: consensus mutated TATA-box probe interaction with nuclear extract (used as negative control); lane 9, 10 and 11: cross-competence with Ts2-CysPrx TATA-box cold probe in a molar excess of 25X, 50X, and 100X, respectively; lane 12: anti-TsTBP1-N antibody without <i>T</i>. <i>solium</i> nuclear extract (negative control). Shifted, super-shifted bands and the free-labeled dsDNA probe, are indicated by arrows. B) Densitometric analysis shows a decrease on the intensity of shifted bands in homologous and heterologous competition. Results are present as percentage mean ± SD of the shifted band in lane 2 (P < 0.005).</p

Molecular Cloning of a cDNA Encoding for <i>Taenia solium</i> TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

<div><p>TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a <i>Taenia solium</i> TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH₂-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. <i>In silico</i> TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in <i>T</i>. <i>solium</i> cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH₂ and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of <i>Taenia crassiceps</i>, an organism close related to <i>T</i>. <i>solium</i>. Electrophoretic mobility shift assays using nuclear extracts from <i>T</i>. <i>solium</i> cysticerci and antibodies against the NH₂-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in <i>T</i>. <i>solium</i> actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.</p></div

EMSA showing the inhibition of the binding of TsTBP1 to TATA-box of pAT5 by the anti-pTsTBP1-C.

<p>Lane 1: labeled TATA-box pAT5 dsDNA-biotin probe without <i>T</i>. <i>solium</i> nuclear extract; lane 2: TATA-box pAT5 interaction with <i>T</i>. <i>solium</i> nuclear extract; lane 3: TATA-box pAT5 plus <i>T</i>. <i>solium</i> nuclear extract and anti-pTsTBP1-C antibodies; lane 4: <i>T</i>. <i>solium</i> nuclear extract plus anti-pTsTBP1-C with TATA-box pAT5; lane 5: TATA-box pAT5 plus <i>T</i>. <i>solium</i> nuclear extract and normal rabbit IgG, lane 6: <i>T</i>. <i>solium</i> nuclear extract plus normal rabbit IgG and TATA box pAT5, lane 7: TATA-box pAT5 plus <i>T</i>. <i>solium</i> nuclear extract and anti-pTsTBP1-N antibodies, and lane 8: <i>T</i>. <i>solium</i> nuclear extract plus anti-pTsTBP1-N antibodies and TATA-box pAT5.</p

Double-stranded DNA probes used for the interaction of TsTBP1 with different TATA-box sequences by EMSA.

<p>In bold letters are represented the putative TATA-box for each gene. Underlined bases are the mutated bases in the TATA-box consensus.</p

Structural analysis of TsTBP1.

<p>A) Ribbon representation of a 3D-homology model for TsTBP1 from the deduced amino acid sequence. It shows a conserved COOH-ter and non-conserved NH₂-ter of TBP structure (β-strands are in yellow, α-helices are in red). B) Superposition of the COOH-ter of TsTBP1 model (in white) with X-ray structure of <i>Saccharomyces cerevisiae</i> TBP1 (in yellow. PDB ID 1RM1), human TFIIB/TBP/DNA complex (in blue, green, and red. PDB ID: 1VOL) and human TFIIA/TBP1 complex (in pink and brown. PDB ID 1NVP). C) Localization in the TsTBP1 model of amino acids involved in DNA recognition (in dark blue) and phosphate groups (gray and cyan). D) Solvent-accessible surface of the COOH-ter TsTBP1 model showed in front and bottom views. The blue patches show the positive density produced by the basic amino acids involved in DNA binding.</p

Composition of nuclear extract proteins and immunodetection of native TsTBP1.

<p>A) 10% SDS-PAGE of cysticerci <i>T</i>. <i>solium</i> nuclear extract patterns stained with Coomassie blue (lane 1). B) Western blot of TsTBP1 on <i>T</i>. <i>solium</i> nuclear extract with: normal serum IgG (lane 1), anti-pTsTBP1-N (lane 2), and anti-pTsTBP1-C antibodies (lane 3). C) Localization of TBP1 on <i>Taenia crassiceps</i> cysticerci sections by confocal microscopy with DAPI (blue), anti-histone H1 (green), anti-pTsTBP1-N antibodies (red) and merging of previous images (yellow signal). Negative control for primary and secondary antibodies, were normal mouse IgG plus anti-mouse IgG-Alexa-568 and normal rabbit IgG plus anti-rabbit IgG-Alexa-488. D) Digital amplification of a single nucleus to observe the localization of DNA (blue), histone H1 (green), and TBP1 (red), and their co-localization (yellow signal).</p