The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element,Biochemistry

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The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element
Biochemistry ( IF 2.9 ) Pub Date : 2020-11-18 , DOI: 10.1021/acs.biochem.0c00571
Samuel Lara-Gonzalez ₁ , Ana Carolina Dantas Machado ₂ , Satyanarayan Rao ₂ , Andrew A Napoli ₁ , Jens Birktoft ₁ , Rosa Di Felice _{2,

3,

4} , Remo Rohs _{2,

3,

5,

6} , Catherine L Lawson _{1,

7}

Affiliation

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A₆-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A₆-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A₆-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to “knock out” binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the “knockout” DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD–DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

中文翻译：

RNA 聚合酶 α 亚基识别上游启动子元件的 DNA 形状

我们在此证明，大肠杆菌RNA 聚合酶 (αCTD)的 α 亚基 C 端结构域通过其特征性的小沟形状和静电势识别上游启动子 (UP) DNA 元件。在两个组成不同的结晶组件中，一对 αCTD 亚基串联结合到长度为 6 bp 的 UP 元件共有 A 束（A ₆束），每个都有其精氨酸 265 胍基团插入小沟。在这些晶体结构以及分别由蒙特卡罗和分子动力学模拟得出的游离和结合 DNA 双链体的计算预测结构中，A ₆束小沟显着变窄。游离A ₆的负静电势-tract DNA 与通用 DNA 相比显着增强。将 A 束缩短 1 bp 显示通过扩大小沟来“敲除”第二个 αCTD 的结合。此外，在 αCTD 中精氨酸 265 突变为丙氨酸的计算得出的结构中，无论有没有“敲除”DNA 突变，与 DNA 的接触都会受到干扰，突出了精氨酸 265 在实现 αCTD-DNA 结合中的重要性。这些结果表明，DNA 形状在 RNA 聚合酶对 DNA 的序列依赖性识别中的重要性与某些转录因子的重要性相当。

更新日期：2020-12-08

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