Global jumping and domain-specific intersegment transfer between DNA cognate sites of the multidomain transcription factor Oct-1
Global jumping and domain-specific intersegment transfer between DNA cognate sites of the multidomain transcription factor Oct-1
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DOI：
10.1073/pnas.0805050105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Clore, G. Marius
中科院分区：
文献类型：
--
作者：
Doucleff, Michaeleen;Clore, G. Marius
在核中发现的高DNA浓度下，DNA结合蛋白通过在单独的DNA链之间跳跃来搜索特定的结合位点。在这里，我们使用N-15（Z） - 交换横向松弛优化了NMR光谱，以表征多域转录因子人类Oct-1的分子间跳跃的机械细节。 OCT-1是：转录因子的POU家族的成员，并包含两个螺旋 - 螺旋 - 螺旋DNA结合域，POUHD和POU，由相对短的柔性链接器连接。发现这两个域以显着不同的速率在特定站点之间进行交换。共转录因子SOX2通过减慢了POUS域的汇率来降低OCT-1> = 5倍和大约20倍的汇率和平衡分离常数。在生理离子强度下测得的DNA依赖性汇率表明，这两个结构域都使用了一个细分段转移机制，该机制不涉及游离蛋白的中介，以及完全解离或跳跃机制在同类位点之间转移。这些数据代表了涉及多域蛋白的蛋白-DNA缔合的域特异性动力学的一个例子，该动力学涉及多域蛋白，并提供了证据，表明细胞分段转移涉及三元中间体或过渡状态，在该状态下，DNA结合域同时桥接了两个不同的DNA片段。
At high DNA concentration, as found in the nucleus, DNA-binding proteins search for specific binding sites by hopping between separate DNA strands. Here, we use N-15(Z)-exchange transverse relaxation optimized NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multidomain transcription factor, human Oct-1. Oct-1 is a member of: the POU family of transcription factors and contains two helix-turn-helix DNA-binding domains, POUHD and POUS, connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The cotranscription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 >= 5-fold and approximate to 20-fold, respectively, by slowing the exchange rate for the POUS domain. DNA-dependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent an example of dissecting domain-specific kinetics for protein-DNA association involving a multidomain protein and provide evidence that intersegmental transfer involves a ternary intermediate, or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously.