首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.
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Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

机译:蛋白酶足迹揭示了转录因子TFIIB上的表面,该表面充当激活剂和共激活剂的界面。

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Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.
机译:模型激活剂GAL4-VP16(由酵母激活剂GAL4的DNA结合结构域和单纯疱疹病毒蛋白VP16的酸性激活结构域组成的嵌合蛋白)对转录的刺激涉及到一系列相互了解的蛋白-蛋白相互作用。 VP16激活域和RNA聚合酶II通用转录机制的组成部分。这些相互作用之一是VP16介导的转录因子TFIIB的结合和募集。然而,这种相互作用需要TATA盒结合蛋白(TBP)相关因子(TAF)或共激活剂才能达到高效的转录复合体装配,据报道,其中一种TAF(果蝇TAF40)与VP16和TFIIB形成三元复合体。 。由于TFIIB在基因激活中的核心作用,我们试图直接可视化这种介导三元复合物形成的蛋白质表面。我们开发了一种称为蛋白酶足迹的方法,其中广泛特异性的胰凝乳蛋白酶和碱性蛋白酶用于探测32P末端标记的TFIIB与GAL4-VP16或TAF40的结合。对切​​割产物的分析揭示了由VP16保护的TFIIB的两个区域免受蛋白酶攻击,其中一个与由TAF40保护的区域重叠。 TFIIB表面上的VP16和TAF40结合位点非常接近,表明该区域可以充当调节界面,介导激活因子和共激活因子对转录复合体装配的影响。

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