Kamine J, Subramanian T, Chinnadurai G
Institute for Molecular Virology, St. Louis University Medical Center, MO 63110.
Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8510-4. doi: 10.1073/pnas.88.19.8510.
The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.
人类免疫缺陷病毒（HIV）编码的Tat蛋白是来自长末端重复序列（LTR）的病毒基因表达的强激活剂。Tat介导的HIV LTR反式激活似乎主要是一个转录事件。据报道，Tat通过与一个称为TAR（反式激活应答元件）的新生RNA靶序列相互作用，在转录起始和延伸水平发挥作用。然而，Tat介导TAR依赖性转录活性的确切机制尚不清楚。为了确定Tat是否通过任何传统的启动子元件与其他真核转录激活剂发挥类似功能，我们测试了Tat在含有结合转录因子Sp1所需的共有序列和TATA盒的合成启动子上的活性。在此，我们报道，通过酵母转录因子GAL4的DNA结合结构域靶向启动子区域的嵌合Tat蛋白激活了合成启动子。由于这种反式激活依赖于Sp1结合位点，Tat显然可以通过与Sp1相互作用介导转录激活。gal4-tat嵌合基因的突变分析表明，Tat的N端48个氨基酸区域构成了Sp1依赖性反式激活的激活区域。Tat的这一区域比Tat的N端58个氨基酸表现出更多的活性，后者包括富含精氨酸的碱性区域。GAL4-Tat的48个氨基酸Tat区域中的特定突变对合成启动子反式激活的影响模拟了这些特定突变对Tat介导的HIV-1 LTR反式激活的影响，表明合成启动子和完整LTR的反式激活通过共同机制发生。
