Allosteric mechanism of induction of CytR-regulated gene expression - CytR repressor-cytidine interaction
Allosteric mechanism of induction of CytR-regulated gene expression - CytR repressor-cytidine interaction
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DOI：
10.1074/jbc.272.27.16962
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发表时间：
1997-07-04
影响因子：
4.8
通讯作者：
Senear, DF
中科院分区：
文献类型：
--
作者：
Barbier, CS;Short, SA;Senear, DF
大肠杆菌CytR调节子的顺反子转录被激活。大肠杆菌cAMP受体蛋白（CRP）的表达，并被elf CRP和CytR组成的多蛋白复合物所抑制。当CytR结合胞苷时，导致去阻遏。CytR是一种同源二聚体，也是LacI家族的成员。所有LacI家族蛋白质的一个中心问题涉及到将配体结合到蛋白质-DNA和调节转录的蛋白质-蛋白质相互作用偶联的变构机制。为了探索CytR的这种机制，我们分析了体外核苷结合及其与CytR与操纵基因DNA的协同结合的偶联。对顺序的胞苷与二聚体CytR结合和CytR与deoP 2的协同结合之间的热力学联系的分析表明，去阻遏仅来自两个胞苷结合步骤之一。通过体内实验验证了这一结论，鉴定出了具有野生型阻遏物功能但胞苷诱导缺陷（CID）的CytR突变体，每个突变体都具有Asp（281）或邻近残基的取代。发现CID CytR 281 N以比野生型CytR低三个数量级的亲和力结合胞苷。发现其他不表现出CID表型的CytR突变体以与野生型CytR相似的亲和力结合胞苷。由一个CytR 281 N和一个野生型亚基组成的异二聚体CytR调节的转录速率与诱导条件下由野生型CytR调节的转录速率进行了比较。数据支持这样的结论：仅第一个胞苷结合步骤就足以诱导。
Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed elf CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member, A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA, Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. go test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified, Each has a substitution for Asp(281) or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were: found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.