DNA Recognition and Wrapping by Escherichia coli RcnR
DNA Recognition and Wrapping by Escherichia coli RcnR
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DOI：
10.1016/j.jmb.2009.08.038
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发表时间：
2009-10-23
影响因子：
5.6
通讯作者：
Chivers, Peter T.
中科院分区：
文献类型：
--
作者：
Iwig, Jeffrey S.;Chivers, Peter T.
大肠杆菌RcnR是最近发现的一个庞大而广泛的细菌转录因子结构家族的创始成员，预计这些转录因子可以对各种环境压力做出反应。RcnR通过协调DNA结合和金属结合活性，直接调节编码RcnA镍钴外排蛋白基因的转录。结核分枝杆菌铜(I)敏感同源物的晶体结构没有揭示该蛋白家族新的全α-螺旋折叠是如何与DNA相互作用的，因为它缺乏一个很好地表征DNA结合的基序。在这项研究中，我们使用等温滴定量热法和足迹技术研究了RcnR-DNA相互作用的生物物理性质。我们发现RcnR四聚体识别一个Tact-G(6)-N-Agta基序，其中两个在rcnA-rcnR基因间隔区。在其他RcnR/CSOR蛋白的许多预测结合部位中都发现了G-链，这里我们证明了它们赋予RcnR操纵子位点A-型DNA特征。有趣的是，RcnR还与核心结合位点两侧类似于50个碱基对的非特异性相互作用，导致DNA包装和单个负超螺旋引入到质粒DNA中。与其他RcnR/CSOR蛋白的比较表明，该家族成员之间的DNA结合可能存在关键差异，这是由于操纵子位点的数量和序列的差异造成的。(C)2009爱思唯尔有限公司。保留所有权利。
Escherichia coli RcnR is a founding member of a recently discovered large and widespread structural family of bacterial transcription factors that are predicted to respond to a variety of environmental stresses. RcnR directly regulates transcription of the gene encoding the RcnA nickel and cobalt efflux protein by coordination of DNA-binding and metal-binding activities. A crystal structure of a Cu(I)-sensing homolog from Mycobacterium tuberculosis did not reveal how the novel all-alpha-helical fold of this protein family interacts with DNA because it lacks a well-characterized DNA-binding motif. In this study, we investigated the biophysical properties of the RcnR-DNA interaction using isothermal titration calorimetry and footprinting techniques. We found that an RcnR tetramer recognizes a TACT-G(6)-N-AGTA motif, of which there are two in the rcnA-rcnR intergenic region. G-tracts are found in many predicted binding sites of other RcnR/CsoR proteins, and here we show that they endow A-form DNA characteristics to the RcnR operator sites. Interestingly, RcnR also interacts nonspecifically with the similar to 50 base pairs flanking the core binding site, resulting in DNA wrapping and the introduction of a single negative supercoil into plasmid DNA. Comparisons with other RcnR/CsoR proteins reveal likely key differences in DNA binding among members of this family that result from variations in the number and sequence of operator sites. (C) 2009 Elsevier Ltd. All rights reserved.