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

Oscar Rodríguez-Lima,Abraham Landa,Lucía Jiménez,Ponciano García-Gutierrez,Ángel Zarain-Herzberg,Roberto Lazzarini,Geoffrey N Gobert

doi:10.1371/journal.pone.0141818

Oscar Rodríguez-Lima, Abraham Landa + Show 5 more

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https://doi.org/10.1371/journal.pone.0141818

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Abstract

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 Taenia solium 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. In silico 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 T. solium 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 Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium 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.

Highlights

Transcription is the process to generate RNA from a gene, and it is carried out by different RNA polymerases
We found two identical cDNA clones of about 900 bp, after screening about 45,000 phages of an adult T. solium cDNA library, which means that TBP1 mRNA correspond to 0.0045% of doi:10.1371/journal.pone.0141818.g004
The amino acid deduced sequence from the cDNA phage clones and its comparison analysis with other TATA-box binding protein (TBP) revealed coding for TBP1 with a predicted molecular weight of 26.7 kDa, which can be divided into an NH2-terminal domain (NH2-ter) and a COOH-ter

Summary

Introduction

Transcription is the process to generate RNA from a gene, and it is carried out by different RNA polymerases. The TATA-box binding protein (TBP) interacts directly with DNA though of this motif, which is typically located at -25 to -35 base pair (bp) relative to the Transcription Start Site (TSS) and has the consensus sequence TATA(A/T)A(A/T) [2, 3]. TBP is an important protein that, together with other general transcription factors (GTFs), forms the pre-initiation complex (PIC) allowing the polymerase to bind the promoter genes and initiate the transcription process. The majority of genes transcribed by all three RNA polymerases lack TATA-box in their promoters; TBP interacts with TBP-associated factors (TAFs) to form the PIC for the RNA Polymerase binding [7,8,9]

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