Van Kaer L, Van Montagu M, Dhaese P
Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium.
J Biol Chem. 1989 Sep 5;264(25):14784-91.
The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties. The active form of repressor appears to be a tetramer. DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF). Three of these sites had been identified earlier as functional operators by genetic analysis. They share a common 14-base pair, asymmetric "core" sequence, 5'-GACGGAAATACAAG-3', termed OR. The three additional sites show significant homology with OR. For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix. Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model. However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction.
枯草芽孢杆菌噬菌体phi 105阻遏蛋白是一种类似λ阻遏蛋白的转录调节蛋白，它在大肠杆菌中过量表达并纯化至近乎同质，以便研究其体外DNA结合特性。阻遏蛋白的活性形式似乎是四聚体。DNase I保护实验表明，阻遏蛋白能特异性结合六个不同的位点，所有这些位点都位于phi 105 EcoRI-F免疫区域（immF）内。其中三个位点先前已通过遗传分析被鉴定为功能性操纵基因。它们共享一个共同的14个碱基对的不对称“核心”序列，5'-GACGGAAATACAAG-3'，称为OR。另外三个位点与OR有显著同源性。对于单个结合位点，羟基自由基足迹分析揭示了通过螺旋一侧建立的对称阻遏蛋白-DNA相互作用。硫酸二甲酯保护表明，保守的OR碱基对位置1、3和4处的鸟嘌呤可能参与与阻遏蛋白的序列特异性相互作用，这与先前提出的识别模型一致。然而，由于OR序列相对于这个GNCG基序不对称，目前仍然难以完全理解这种相互作用的分子基础。
