Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.
Mol Cell Biol. 2012 Dec;32(23):4780-93. doi: 10.1128/MCB.00206-12. Epub 2012 Sep 24.
Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription.
转录因子通过在转录周期的多个步骤中组装和重塑复合物来调节真核 RNA 聚合酶 II (Pol II) 的活性。在 HIV 中，我们之前提出了一个两步模型，即病毒 Tat 蛋白首先与无活性的 P-TEFb：7SK snRNP 复合物在启动子上预组装，然后将 P-TEFb 转移到新生转录本上的 TAR 上，取代抑制性 snRNP，导致 Pol II 磷酸化并刺激延伸。尚不清楚 Tat：P-TEFb 复合物如何转移到 TAR 以激活 P-TEFb 激酶。在这里，我们表明，人工招募到新生转录本上的 P-TEFb 不能胜任转录，而是由于其与 7SK snRNP 的组装而仍然处于无活性状态。转染的 Tat 能够从 snRNP 上置换激酶抑制剂 Hexim1 并激活 P-TEFb，从而将 Tat 对激酶激活和 TAR 结合的要求解耦。通过将 Tat 的全面突变与多种基于细胞的报告基因测定结合使用，这些测定探针 Tat 在不同排列中的活性，我们从遗传上定义了一个过渡步骤，其中预组装的 Tat：P-TEFb 复合物切换到 TAR。我们提出，Tat 中保守的残基网络已经进化到控制这种转变，从而将宿主延伸机制切换到病毒转录。
