Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA.
Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA.
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重组 HIV-1 Tat 的表征及其与 TAR RNA 的相互作用。
DOI：
10.1021/bi00163a014
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Nalin,CM
中科院分区：
文献类型：
--
作者：
Slice,LW;Codner,E;Antelman,D;Holly,M;Wegrzynski,B;Wang,J;Toome,V;Hsu,MC;Nalin,CM
1992年8月17日收到的修订稿摘要：重组HIV-1 TAT(TAT 1-86)已从大肠杆菌的细胞质部分中纯化出来，不使用蛋白质变性剂或杂交剂。氯喹介导的纯化蛋白被细胞摄取导致反式激活。人类免疫缺陷病毒LTR启动子。原子吸收光谱分析表明，TAT保留了1.64摩尔的锌离子/摩尔蛋白质。圆二色谱测量表明，重组TAT的结构中含有15-20%的α-螺旋。滤膜结合分析表明，在25░C，0.05M离子强度，pH 7.5的条件下，TAT与63个核苷酸的靶TAR RNA结合，解离常数(AD)为10 nM，其化学计量比为1：1。非静电相互作用提供了自由缔合能的主要来源。最适pH值出现在较宽的H+浓度范围内，而Ka的盐依赖性表明形成了单离子对。紫外线诱导的蛋白质-RNA交联物产生标记的TAT-TAR RNA加合物，表明TAT蛋白与TAR RNA发生了直接接触。人类免疫缺陷病毒1型TAT蛋白是一种在感染细胞中表达的病毒编码蛋白。这种蛋白质对病毒复制是必不可少的(Arya等人，1985；Sodroski等人，1985；Fisher等人，1986；Dayton等人，1986；Sadai等人，1988)。TAT是HIV-1 LTR启动子的转录反式激活因子，它含有转录起始点下游的顺式反应序列TAR(Cullen，1986；Peterlin等人，1986；Rice&Mathews，1988；Jakobovits等人，1988；冯&Holland，1988；Hauber&Cullen，1988)。该蛋白上调HIV-1病毒的表达
Revised Manuscript Received August 17, 1992 abstract: Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fractionof Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation. of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicatedthat the structure of recombinant Tat contains 15-20% a-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Ad) of 10 nM at 25░ C, 0.05 M ionic strength, pH 7.5, in a 1: 1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Ka indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.The Tat protein of human immunodeficiency virus (HIV) 1 type 1 is a virally encoded protein expressed in infected cells. The protein is essential for viral replication (Arya et al., 1985; Sodroski et al., 1985; Fisher et al., 1986; Dayton et al., 1986; Sadai et al., 1988). Tat is a transcription transactivator for the HIV-1 LTR promoter which contains the cis-acting responsive sequence TAR downstream from the transcription start site (Cullen, 1986; Peterlin et al., 1986; Rice & Mathews, 1988; Jakobovits et al., 1988; Feng & Holland, 1988; Hauber & Cullen, 1988). The protein up-regulates HIV-1viral