Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunits
Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunits
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DOI：
10.1074/jbc.m002511200
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Severinov, K
中科院分区：
文献类型：
--
作者：
Nechaev, S;Chlenov, M;Severinov, K
从大肠杆菌RNA聚合酶的β亚基缺失10个进化上保守的氨基酸导致不能有效地保持在DNA上的突变酶。由突变酶形成的开放启动子复合物与封闭复合物快速平衡，并且与野生型复合物不同，对DNA竞争物肝素高度敏感（Martin，E.，Sagitov，V.，Burova，E.，Nikiforov，V，和Goldfarb，A（1992）J，Biol，Chem.267，20175-20180），在此我们表明，尽管存在这种不稳定性，但当野生型复合物完全闭合时，突变酶在低至0 ℃的温度下形成部分开放的复合物。因此，开放启动子复合物的两个标志，即对DNA竞争者挑战的稳定性和对低温的敏感性，可以通过突变解偶联，并且在野生型复合物中可能是独立的。我们使用水生栖热菌RNA聚合酶核心的高分辨率结构来建立一个启动子复合物形成的功能模型，该模型解释了所观察到的E. coli RNA聚合酶突变体。
Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA, Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E,, Sagitov, V,, Burova, E., Nikiforov, V,, and Goldfarb, A (1992) J, Biol, Chem. 267, 20175-20180), Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.