Institut für Biologie, Humboldt Universität zu Berlin, AG Bakteriengenetik, Chausseestrasse 117, 10115 Berlin, Germany.
Mol Genet Genomics. 2012 Feb;287(2):111-22. doi: 10.1007/s00438-011-0666-4. Epub 2011 Dec 20.
AbrB is a global regulator of transition state that is known to repress more than 100 genes in Bacillus species. Although AbrB is involved in the regulation of most cellular processes, a conserved binding motif seems to be elusive. Thus, the mechanism of AbrB-mediated transcriptional control is still unclear. In our previous work we identified two separate AbrB-binding sites within phytase gene region (phyC) of Bacillus amyloliquefaciens FZB45, whose integrity is essential for repression. Comparable architecture of AbrB-binding sites is also described for tycA that encodes an antibiotic synthesis enzyme. Considering the size of the AbrB tetramer (56 kDa) and other AbrB binding motifs (~20 to 98 bp) we hypothesized preferred binding positions within both AbrB sites of phyC that exhibit higher affinities to AbrB. Thus, we used surface plasmon resonance (SPR) to study the binding kinetics between AbrB and 40-bp ds-oligonucleotides that were derived from both binding sites. Surface plasmon resonance sensorgrams revealed strong binding kinetics that showed nearly no dissociation and positive cooperativity of the AbrB-DNA interaction to the whole AbrB-binding site 2 and to a small part of AbrB-binding site 1. Using chemically modified DNA we found bases contacting AbrB mainly at one face of the DNA-helix within a core region separated by one helical turn each. High content of modified guanines presented in the control reaction of the KMnO(4) interference assay indicated distortion of the DNA-structure of phyC. In vitro transcription assays and base substitutions within the core region support this idea and the cooperativity of AbrB binding.
AbrB 是一种过渡状态的全局调节剂，已知它可以抑制芽孢杆菌属中的 100 多个基因。虽然 AbrB 参与了大多数细胞过程的调节，但似乎没有保守的结合基序。因此，AbrB 介导的转录控制机制尚不清楚。在我们之前的工作中，我们在解磷菌（Bacillus amyloliquefaciens FZB45）的植酸酶基因区域（phyC）中鉴定了两个独立的 AbrB 结合位点，其完整性对于抑制是必不可少的。类似的 AbrB 结合位点结构也在编码抗生素合成酶的 tycA 中进行了描述。考虑到 AbrB 四聚体（56 kDa）的大小和其他 AbrB 结合基序（~20 至 98 bp），我们假设在 phyC 的两个 AbrB 结合位点内存在优先结合位置，这些位置与 AbrB 具有更高的亲和力。因此，我们使用表面等离子体共振（SPR）研究了 AbrB 与源自两个结合位点的 40-bp ds-寡核苷酸之间的结合动力学。表面等离子体共振传感器图谱显示出强烈的结合动力学，几乎没有解离，并且 AbrB-DNA 相互作用对整个 AbrB 结合位点 2 和 AbrB 结合位点 1 的一小部分具有正协同性。使用化学修饰的 DNA，我们发现与 AbrB 接触的碱基主要位于 DNA 螺旋的一个面上，每个螺旋旋转一次分离。在 KMnO4 干扰测定的对照反应中呈现的高含量修饰鸟嘌呤表明 phyC 的 DNA 结构发生了扭曲。体外转录测定和核心区域内的碱基取代支持了这一观点以及 AbrB 结合的协同性。
