Rodríguez-Lima Oscar, García-Gutierrez Ponciano, Jiménez Lucía, Zarain-Herzberg Ángel, Lazzarini Roberto, Landa Abraham
Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F., México.
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, México D.F., México.
PLoS One. 2015 Nov 3;10(11):e0141818. doi: 10.1371/journal.pone.0141818. eCollection 2015.
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.
TATA 框结合蛋白（TBP）是 TATA 框和无 TATA 框基因启动子的一种重要调控转录因子。我们报告了一个全长 cDNA 的克隆和表征，该 cDNA 编码猪带绦虫 TATA 框结合蛋白 1（TsTBP1）。从其核苷酸序列推导的氨基酸组成显示，它编码一个 238 个残基的蛋白质，预测分子量为 26.7 kDa，理论 pI 为 10.6。与猪和人 TBP1 相比，其 NH2 末端结构域没有保守性。然而，它在大小和氨基酸同一性上与带绦虫 TBP1 高度保守。相比之下，TsTBP1 的 COOH 末端结构域在生物之间高度保守，并包含与 TATA 框以及 TFIIA 和 TFIIB 相互作用所涉及的氨基酸。在计算机模拟中，TsTBP1 建模显示 COOH 末端结构域形成了 TBP 家族的经典鞍形结构，末端有一个α螺旋，这在猪和人中不存在。使用针对 TsTBP1 的 NH2 和 COOH 末端结构域中的两个合成肽产生的兔抗体，通过蛋白质印迹法在猪带绦虫囊尾蚴的核提取物中检测到天然 TsTBP1。这些抗体通过免疫荧光技术，在形成粗颈带绦虫囊尾蚴膀胱壁的细胞核中鉴定出 TBP1，粗颈带绦虫是一种与猪带绦虫密切相关的生物。使用猪带绦虫囊尾蚴的核提取物和针对 TsTBP1 的 NH2 末端结构域的抗体进行电泳迁移率变动分析，显示天然 TsTBP1 与猪带绦虫肌动蛋白 5（pAT5）和 2 - 半胱氨酸过氧化物酶（Ts2 - CysPrx）基因启动子中存在的 TATA 框相互作用；相反，当使用针对 TsTBP1 的抗 COOH 末端结构域的抗体时，它们抑制了 TsTBP1 与 pAT5 启动子基因的 TATA 框的结合。
