The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA Polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and core.
The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA Polymerase more rapidly in the absence of sigma70 region 1.1: evidence that region 1.1 stabilizes the interaction between sigma70 and core.
复制标题
在没有 sigma70 区域 1.1 的情况下，噬菌体 T4 抑制剂和共激活剂 AsiA 可以更快地抑制大肠杆菌 RNA 聚合酶：证据表明区域 1.1 稳定了 sigma70 和核心之间的相互作用。
DOI：
10.1128/jb.188.4.1279-1285.2006
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发表时间：
2006
影响因子：
3.2
通讯作者：
Mulamba,Rebecca
中科院分区：
文献类型：
--
作者：
Hinton,DeborahM;Vuthoori,Srilatha;Mulamba,Rebecca
大肠杆菌RNA聚合酶的主要σ亚基σ70的N端区域（区域1.1）是一个带负电荷的结构域，影响σ 70区域2和4的DNA结合特性。区域1.1阻止游离σ 70与DNA的相互作用，并调节在某些启动子处稳定（开放）聚合酶/启动子复合物的形成。噬菌体T4 AsiA蛋白是一种σ70依赖性转录的抑制剂，其启动子需要σ 70区域4和−35 DNA元件之间的相互作用，并且是T4 MotA依赖性启动子转录的共激活因子。与AsiA一样，T4激活剂MotA也与σ 70区域4相互作用。我们研究了区域1.1对AsiA抑制和MotA/AsiA激活的影响。我们发现，σ 70区域1.1不是T4中间启动子PuvsX激活MotA/AsiA所必需的。然而，当区域1.1缺失时，聚合酶的AsiA抑制率和MotA/AsiA活化率显著增加。我们还发现用缺少1.1区的σ 70重组的RNA聚合酶比用全长σ70重组的聚合酶稳定性差。我们以前的工作已经证明，当AsiA与游离σ 70的第4区结合，然后AsiA/σ 70复合物与核心结合时，形成AsiA抑制的聚合酶。我们的结果表明，在区域1.1的情况下，聚合酶全酶和游离σ 70+核心之间的动态平衡发生了变化，在任何给定的时间产生更多的游离σ 70。因此，当RNA聚合酶缺乏区域1.1时，AsiA抑制和AsiA/MotA激活的速率增加，因为游离σ70的可用性增加。以前的工作都认为和反对区域1.1和4之间的直接相互作用。使用一个E。colitwo-杂交测定，我们没有检测到这些区域之间的相互作用。这一结果支持了区域1.1阻止游离σ 70与DNA结合的能力是通过间接作用产生的观点。
The N-terminal region (region 1.1) of σ70, the primary σ subunit ofEscherichia coliRNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70regions 2 and 4. Region 1.1 prevents the interaction of free σ70with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70and then the AsiA/σ70complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70plus core, yielding more free σ70at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using anE. colitwo-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70arises through an indirect effect.