Role of protein-protein bridging interactions on cooperative assembly of DNA-bound CRP-CytR-CRP complex and regulation of the Escherichia coli CytR regulon
Role of protein-protein bridging interactions on cooperative assembly of DNA-bound CRP-CytR-CRP complex and regulation of the Escherichia coli CytR regulon
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DOI：
10.1021/bi0271143
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发表时间：
2003-04-08
期刊：
影响因子：
2.9
通讯作者：
Senear, DF
中科院分区：
文献类型：
--
作者：
Chahla, M;Wooll, J;Senear, DF
组成大肠杆菌CytR调节子的未连接的操纵子通过两种基因调节蛋白（cAMP受体蛋白（CRP）和胞苷阻遏物（CytR））之间的相互作用协调控制。CytR控制CRP介导的RNA聚合酶的募集和激活与转录抑制之间的平衡。当CytR与位于CytR调节启动子上游的操纵子（CytO）结合时，CytR与CRP与侧翼串联启动子结合时，两者之间的协同相互作用对CytR的调节作用至关重要。当CytR结合胞苷时，协同性降低，导致转录活性增加。然而，这种胞苷介导的效应在启动子之间变化，表明胞苷与CytR结合和CytR-CRP结合之间的偶联对启动子结构敏感。为了研究这些效应的化学和结构基础，我们研究了胞苷结合如何影响溶液中CytR和CRP之间的结合，以及它如何影响缺乏DNA结合HTH结构域的CytR缺失突变体与与udpP或deoP 2结合的串联CRP二聚体的结合。缺失突变体，我们在这里显示，保留变构，诱导剂结合结构域的天然功能，但不结合DNA的表达和纯化。我们将这些称为核心域。尽管在溶液中CytR和CRP之间仅存在弱关联，但我们的结果表明形成了相对稳定的复合物，其中核心结构域在与udpP或deoP 2结合时在串联CRP二聚体之间形成蛋白质桥。桥络合物形成的Δ G度约为-7.8kcal/mol。这远远超过了考虑协同性所需的量（-2.5至-3千卡/摩尔）。桥复合物被胞苷结合显著地去稳定化，并且在两种启动子复合物中达到相同的程度（Δ Δ G度近似于+2kcal/mol）。即使有这种不稳定性，由胞苷配体的核心结构域形成桥复合物的Δ G度本身仍然足以解释协同性。这些发现表明，胞苷结合CytR和CytR-CRP协会之间的直接耦合不占启动子特异性的协同效应。相反，胞苷结合必须诱导更刚性或以某种其他方式对不同启动子之间操纵位点几何排列变化的耐受性较差的CytR构象。
The unlinked operons that comprise the Escherichia coli CytR regulon are controlled coordinately through interactions between two gene regulatory proteins, the cAMP receptor protein (CRP) and the cytidine repressor (CytR). CytR controls the balance between CRP-mediated recruitment and activation of RNA polymerase and transcriptional repression. Cooperative interactions between CytR, when bound to an operator (CytO) located upstream of a CytR-regulated promoter, and CRP, when bound to flanking tandem promoters, are critical to the regulatory role of CytR. When CytR binds cytidine, cooperativity is reduced resulting in increased transcriptional activity. However, this cytidine-mediated effect varies among promoters, suggesting that coupling between cytidine binding to CytR and CytR-CRP association is sensitive to promoter structure. To investigate the chemical and structural basis for these effects, we investigated how cytidine binding affects association between CytR and CRP in solution and how it affects the binding of CytR deletion mutants lacking the DNA binding HTH domain, with tandem CRP dimers bound to either udpP or deoP2. Deletion mutants that, as we show here, retain the native functions of the allosteric, inducer-binding domain but do not bind DNA were expressed and purified. We refer to these as Core domain. Despite only weak association between CytR and CRP in solution, our results demonstrate the formation of a relatively stable complex in which the Core domain forms a protein bridge between tandem CRP dimers when bound to either udpP or deoP2. The DeltaGdegrees for bridge complex formation is about -7.8 kcal/mol. This is well in excess of that required to account for cooperativity (-2.5 to -3 kcal/mol). The bridge complexes are significantly destabilized by cytidine binding, and to the same extent in both promoter complexes (DeltaDeltaGdegrees approximate to +2 kcal/mol). Even with this destabilization, DeltaGdegrees for bridge complex formation by cytidine-liganded Core domain is still sufficient by itself to account for cooperativity. These findings demonstrate that direct coupling between cytidine binding to CytR and CytR-CRP association does not account for promoter-specific effects on cooperativity. Instead, cytidine binding must induce a CytR conformation that is more rigid or in some other way less tolerant of the variation in the geometric arrangement of operator sites between different promoters.