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首页> 外文期刊>Journal of bacteriology >Burkholderia xenovorans RcoMBx-1, a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide
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Burkholderia xenovorans RcoMBx-1, a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide

机译：伯克霍尔德菌xcovorans RcoMBx-1，一种转录调节系统，用于感知低水平和持久性一氧化碳水平

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The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal “LytTR” DNA-binding domain. Here the RcoMBx-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (～10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ～60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5′-TTnnnG-3′) that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved “TTnnnG” motif residues and their triplet arrangement and were also used to determine an improved binding motif [5′-CnnC(C/A)(G/A)TTCAnG-3′] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoMBx-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoMBx-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional “accumulating switch” that senses low but persistent CO levels.
机译：单组分RcoM转录因子将N末端结合的血红素辅因子与C末端“ LytTR” DNA结合结构域偶联。异种表达伯克霍尔德氏菌LB400的RcoM Bx -1蛋白，然后以结合CO最少（〜10％）的形式纯化，发现与该结合稳定具有纳摩尔亲和力的效应子。 DNase I保护试验表明，CO相关形式以微摩尔亲和力与两个〜60 bp的DNA区域结合，每个区域由一组新颖的三个直接重复结合位点组成，这些结合位点的中心相距21 bp。与每个区域的结合是独立的，而与区域内三联体结合位点的结合则是协同的，取决于间隔和序列，并且以阶段性​​DNase I过度活跃和保护模式为标志，与核蛋白复合物的DNA构象的显着变化相一致。每个受保护的结合位点跨越一个保守的基序（5'-TTnnnG-3'），该基序一式三份出现在十几个生物体的推定RcoM结合区域中。 体内筛选证实了保守的“ TTnnnG”基序残基及其三联体排列的功能重要性，还用于确定改进的结合基序[5'-CnnC（C / A）（G / A ）TTCAnG-3']与标准LytTR结构域/ DNA结合位点更为接近。在体外证明了RcoM Bx -1与多种DNA探针的低亲和力和CO依赖性结合。我们认为，对于RcoM Bx -1蛋白，高CO亲和力与多个低亲和力DNA结合事件结合在一起构成了转录“积累开关”，其感觉很低，但持续的一氧化碳水平。

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