HIV TAT GENE AND TRANSCRIPTIONAL ACTIVATION

HIV Tat蛋白是HIV转录的有效反式激活因子， 增加了RNA的起始和延伸。纯净型全长86 氨基酸Tat蛋白在体外特异性反式激活HIV LTR 25到50倍。HIV转录在不同的功能步骤中被阻断 在起始期和延伸期形成复合体。类似于 对照AdML启动子，HIV碱基起始复合体组装敏感 在添加核苷之前添加0.015%肌糖 三磷酸盐。对0.05%肌糖的抗性需要添加G， C和U，构成HIV RNA转录本的前13个碱基。 在体外转录反应中加入TAT可以缓解 0.015%肌糖阻滞剂。这些TAT诱导的复合体对 0.05%肌糖，表明转录启动没有 发生了。与这一假设一致，G、C和U的添加 TAT诱导的转录复合体允许快速转化为 转录起始复合体。TAT还促进了 重组转录系统中的起始复合体 部分纯化的转录因子和聚合酶II。 形成稳定的引发络合物，TAT提高了反应速度和反应速度。 转录延伸效率。这些结果表明，TAT， 除了它在RNA延伸中已被证明的作用外，还促进了 体外转录启动。 与这些观察结果一致的是，TAT直接与基底结合 转录因子TFIID。HeLa提取物的转录活性 在TAT亲和柱层析后被耗尽， 特别保留了TFIID。TFIID无法绑定到突变TAT 含有单一氨基酸取代的亲和层析柱。塔塔 在大肠杆菌中表达的结合蛋白(TBP)与TAT结合，表明 TAT与TFIID的基本亚基相互作用。多肽竞赛和 TFIID结合分析表明，TFIID的保守核心区 TAT蛋白介导TAT-TFIID相互作用。

The HIV Tat protein is a potent transactivator of HIV transcription, increasing both RNA initiation and elongation. Purified, full-length 86 amino acid Tat protein specifically transactivates the HIV LTR in vitro 25- to 50-fold. HIV transcription was blocked at various functional steps during initiation and elongation complex formation. Similar to the control AdML promoter, HIV basal initiation complex assembly was sensitive to the addition of 0.015% sarkosyl prior to the addition of nucleoside triphosphates. Resistance to 0.05% sarkosyl required the addition of G, C and U which constitute the first 13 bases of the HIV RNA transcript. The addition of Tat to the in vitro transcription reaction relieved the 0.015% sarkosyl block. These Tat-induced complexes were sensitive to 0.05% sarkosyl, suggesting that transcriptional initiation had not occurred. Consistent with this hypothesis, the addition of G, C and U to the Tat-induced transcription complexes allowed the rapid conversion to transcription initiation complexes. Tat also facilitated the formation of initiation complexes in a reconstituted transcription system containing partially purified transcription factors and polymerase II. Following the formation of stable initiation complexes, Tat increased the rate and efficiency of transcription elongation. These results suggest that Tat, in addition to its demonstrated role in RNA elongation, facilitates transcription initiation in vitro. Consistent with these observations, Tat binds directly to the basal transcription factor TFIID. The transcriptional activity of HeLa extracts was depleted after chromatography on Tat affinity columns, which specifically retained TFIID. TFIID failed to bind to a mutant Tat affinity column which contained a single amino acid substitution. TATA binding protein (TBP), expressed in E. coli, binds to Tat, suggesting that Tat interacts with the basic subunit of TFIID. Peptide competition and TFIID binding analysis demonstrates that the conserved core region of the Tat protein mediates the Tat-TFIID interaction.