Identification of a minimal binding element within the T7 RNA polymerase promoter
Identification of a minimal binding element within the T7 RNA polymerase promoter
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DOI：
10.1006/jmbi.1997.1350
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发表时间：
1997-11-07
影响因子：
5.6
通讯作者：
Martin, CT
中科院分区：
文献类型：
--
作者：
Ujvari, A;Martin, CT
已经提出T7 RNA聚合酶启动子包含两个结构域：位置-5上游的结合区域通过明显传统的双链体接触识别，而位置-5下游的催化结构域以解链构型结合。该模型通过以下聚合酶与代表共有启动子序列截短的一系列合成寡核苷酸的结合来测试。连接到启动子的上游端的罗丹明染料的荧光各向异性的增加在简单的热力学滴定中提供了酶结合的非常灵敏的测量，并且允许测定解离常数的增加和减少。天然启动子的最佳拟合值K-d = 4.0 nM与先前的荧光和稳态测量结果非常一致。缺失下游DNA至位置-1或至位置-5导致结合增加5倍，而进一步连续的上游单碱基缺失导致结合减少20和500倍。这些结果表明，启动子上游的（双链体）区域（包括位置-5）对于紧密结合是必需的和足够的，并且代表启动子的核心结合元件。我们提出了一个模型，其中T7 RNA聚合酶使用上游结合能的一部分来熔化启动子的下游起始区。我们还表明，镁的存在是必要的最佳结合，但不是特定的酶-启动子复合物的形成，我们建议，镁是不需要熔化的启动子。(C)出版社：Academic Press Limited。
The T7 RNA polymerase promoter has been proposed to contain two domains: the binding region-upstream of position -5 is recognized through apparently traditional duplex contacts, while the catalytic domain downstream of position -5 is bound in a melted configuration. This model is tested by following polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence. The increase in the fluorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations, and allows the determination of both increases and decreases in the dissociation constant. The best fit value of K-d = 4.0 nM for the native promoter is in good agreement with previous fluorescence and steady state measurements. Deletion of the downstream DNA up to position -1 or to position -5 leads to a fivefold increase in binding, while further sequential single-base deletions upstream result in 20 and 500-fold decreases in binding. These results indicate that the (duplex) region of the promoter upstream of and including position -5 is both necessary and sufficient for tight binding, and represents the core binding element of the promoter. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. We also show that the presence of magnesium is necessary for optimal binding, but not for specific enzyme-promoter complex formation, and we propose that magnesium is not required for melting of the promoter. (C) 1997 Academic Press Limited.