AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter.
AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter.
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DOI：
10.7554/elife.02375
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发表时间：
2014-04-24
期刊：
影响因子：
7.7
通讯作者：
Alber T
中科院分区：
文献类型：
--
作者：
Schulze-Gahmen U;Lu H;Zhou Q;Alber T
超长复合体(SECs)是许多人类基因转录延长所必需的，包括整合的HIV-1基因组。在HIV-1启动子处，病毒Tat蛋白在一秒内同时与新生的TAR RNA和P-TEFb激酶的CycT1亚基结合。为了了解TAT和TAR对SEC的优先招募，我们测定了由TAT、P-TEFb和SEC支架AFF4组成的四元络合物的晶体结构。TAT和AFF4在CycT1表面折叠，直接相互作用。AFF4同源基因AFF1中的界面突变降低了体内TAT-AFF1亲和力和HIV启动子的TAT依赖转录。在TAT存在下，AFF4结合部分地排列了CycT1的TAT-TAR识别基序，并使TAT-P-TEFb对TAR的亲和力增加了30倍。这些研究表明，与P-TEFb相比，AFF4起到了两步过滤的作用，提高了SEC对TAT和焦油的选择性。DOI：http://dx.doi.org/10.7554/eLife.02375.001许多基因以蛋白质的形式表达的速度取决于一种称为转录延伸的过程。这个过程发生在定义基因的DNA区域被转录成RNA分子时，它被一种名为RNA聚合酶II的酶催化。然而，这个过程通常在启动后不久就停止了，需要另一种名为正转录延伸因子的酶来重新启动它。人类免疫缺陷病毒(HIV)是一种逆转录病毒，它劫持免疫细胞内的基因表达机制，以便自我复制。为了尽可能有效地做到这一点，延伸因子需要尽快重新启动转录过程。为了确保这种情况发生，病毒会产生一种名为TAT的蛋白质，它会与已经合成的一小段RNA结合。与此同时，Tat蛋白质还与其他蛋白质结合，形成一个称为超级延长复合体的多蛋白质机器。超级伸长复合体中的其他蛋白质包括一种名为AFF4的“支架”蛋白，一种名为P-TEFb的正伸长因子，以及至少两种额外的转录因子。直到最近，研究人员还不知道TAT蛋白如何能够在不招募包含类似蛋白质亚单位的其他复合体的情况下，将超伸长复合体招募到正确的位置。现在，Schulze-Gahmen等人。通过研究延伸因子P-TEFb与TAT蛋白和名为AFF4的支架蛋白形成复合体时形成的复合体的晶体结构，揭示了这一谜团。结果表明，TAT与支架蛋白之间的直接相互作用有助于将超伸长复合体招募到正确的位置。TAT、AFF4和P-TEFb之间的三向相互作用形成了一个结合表面，鼓励复合体与RNA结合。总体而言，舒尔茨-加赫曼等人。结果表明，超延伸复合体更有可能被TAT蛋白质识别，然后与RNA结合，而不仅仅是延伸因子本身。DOI：http://dx.doi.org/10.7554/eLife.02375.002
Superelongation complexes (SECs) are essential for transcription elongation of many human genes, including the integrated HIV-1 genome. At the HIV-1 promoter, the viral Tat protein binds simultaneously to the nascent TAR RNA and the CycT1 subunit of the P-TEFb kinase in a SEC. To understand the preferential recruitment of SECs by Tat and TAR, we determined the crystal structure of a quaternary complex containing Tat, P-TEFb, and the SEC scaffold, AFF4. Tat and AFF4 fold on the surface of CycT1 and interact directly. Interface mutations in the AFF4 homolog AFF1 reduced Tat–AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 binding in the presence of Tat partially orders the CycT1 Tat–TAR recognition motif and increases the affinity of Tat-P-TEFb for TAR 30-fold. These studies indicate that AFF4 acts as a two-step filter to increase the selectivity of Tat and TAR for SECs over P-TEFb alone. DOI: http://dx.doi.org/10.7554/eLife.02375.001 The rate at which many genes are expressed as proteins depends on a process called transcriptional elongation. This process takes place as the region of DNA that defines the gene is transcribed into an RNA molecule, and it is catalyzed by an enzyme called RNA polymerase II. However, this process often stalls shortly after it starts, and another enzyme called a positive transcription elongation factor is needed to restart it. The human immunodeficiency virus (HIV) is a retrovirus that hijacks the gene expression machinery inside immune cells in order to replicate itself. To do this as efficiently as possible, the elongation factor needs to restart the transcription process as quickly as possible. To ensure that this happens the virus produces a protein called Tat that binds to the short region of RNA that has already been made. At the same time the Tat protein also combines with other proteins to form a multi-protein machine called the super elongation complex. Other proteins in the super elongation complex include a ‘scaffold’ protein called AFF4, a positive elongation factor called P-TEFb, and at least two additional transcription factors. Until recently researchers did not know how the Tat protein was able to recruit super elongation complexes to the correct location without recruiting other complexes that contained similar protein subunits. Now Schulze-Gahmen et al. have shed new light on this mystery by working out the crystal structure of the complex formed by the elongation factor P-TEFb when it forms a complex with the Tat protein and a scaffold protein called AFF4. The results show that direct interactions between the Tat and scaffold proteins help to recruit the super elongation complex to the correct location. The three-way interactions between Tat, AFF4, and P-TEFb form a binding surface that encourages the complex to bind to the RNA. Overall, Schulze-Gahmen et al. show that the super elongation complex is much more likely to be recognized by the Tat protein and then bind to RNA than just the elongation factor on its own. DOI: http://dx.doi.org/10.7554/eLife.02375.002