Caron C, Rousset R, Béraud C, Moncollin V, Egly J M, Jalinot P
Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon, UMR 49, CNRS, France.
EMBO J. 1993 Nov;12(11):4269-78. doi: 10.1002/j.1460-2075.1993.tb06111.x.
The human T-cell leukemia virus type I (HTLV-I) codes for the potent transcriptional activator, Tax1, which induces the enhancer activity of various enhancer elements. In the case of the 21 bp enhancer of the HTLV-I provirus, this induction is correlated with the association of Tax1 with this DNA element via a specific cellular factor. That the indirect association of Tax1 with DNA can lead to transcriptional activation has also been supported by the study of chimeric GAL4-Tax1 proteins. The GAL4-Tax1 stimulatory effect exhibits a strong self-squelching. In order to determine whether Tax1 interacts directly with the general transcription factors or via intermediary molecules, we have analyzed how overexpression of the TATA binding protein (TBP) and TFIIB protein affects the squelching curve of GAL4-Tax1. The data presented here show that overexpression of TBP strongly increases the stimulatory effect of GAL4-Tax1, causes a displacement of the maximum of the squelching curve and partially alleviates the squelching. Under similar conditions TFIIB exhibited little effect. From these results we conclude that Tax1 can increase the recruitment of TBP by directly interacting with this protein. Biochemical experiments with purified proteins produced in bacteria confirmed that Tax1 can interact with TBP but not with TFIIB. Tax1 interacts with the conserved C-terminal part of TBP. Analysis of the ability of different mutants of Tax1 fused to the GAL4 DNA binding domain to activate transcription and to associate with TBP, showed that these activities are correlated. However, since one transcriptionally inactive mutant was able to interact efficiently with TBP in vitro, it would appear that an event other than the Tax1-TBP contact also intervenes in the activation of transcription by Tax1.
人类I型T细胞白血病病毒（HTLV-I）编码强效转录激活因子Tax1，该因子可诱导多种增强子元件的增强子活性。就HTLV-I前病毒的21bp增强子而言，这种诱导作用与Tax1通过特定细胞因子与该DNA元件的结合相关。对嵌合GAL4-Tax1蛋白的研究也支持了Tax1与DNA的间接结合可导致转录激活这一观点。GAL4-Tax1的刺激作用表现出强烈的自我抑制。为了确定Tax1是直接与通用转录因子相互作用还是通过中间分子相互作用，我们分析了TATA结合蛋白（TBP）和TFIIB蛋白的过表达如何影响GAL4-Tax1的抑制曲线。此处呈现的数据表明，TBP的过表达强烈增强了GAL4-Tax1的刺激作用，导致抑制曲线最大值的位移，并部分缓解了抑制作用。在类似条件下，TFIIB几乎没有影响。从这些结果我们得出结论，Tax1可通过直接与该蛋白相互作用来增加TBP的募集。用细菌中产生的纯化蛋白进行的生化实验证实，Tax1可与TBP相互作用，但不能与TFIIB相互作用。Tax1与TBP保守的C末端部分相互作用。对与GAL4 DNA结合域融合的Tax1不同突变体激活转录及与TBP结合能力的分析表明，这些活性是相关的。然而，由于一个转录无活性的突变体在体外能够有效地与TBP相互作用，因此似乎除了Tax1-TBP接触之外的其他事件也参与了Tax1对转录的激活。
