Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage
Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage
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志贺毒素编码噬菌体 933W 阻遏蛋白的纯化和表征：DNA 结合、基因调控和自动裂解
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通讯作者：
S. Buranen
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作者：
C. Y. Lee;S. Buranen
志贺毒素(STX)是志贺毒素编码的大肠杆菌(STEC)菌株的主要毒力因子，其编码基因存在于所有已知STEC菌株中的Lambdoid噬菌体上。STX基因只在这些温带噬菌体的裂解生长过程中表达。我们克隆了志贺毒素编码噬菌体933W抑制子的基因，并检测了高表达、纯化的fi蛋白的DNA结合和转录调节活性。933W的抑制子与933W的右操纵子(O-R)中的三个位点结合，是几乎所有噬菌体抑制子的典型特征。同样典型的是，当与O-R结合时，933W抑制物在P-RM启动子上起激活作用，在P-R启动子上起抑制作用。与其他噬菌体相比，933W左侧操纵子(O L)只含有两个阻遏因子结合位点，但O L结合的阻遏因子fi仍能有效抑制P L的转录。Lambdoid噬菌体的诱导需要激活抑制子的DNA结合活性。在到目前为止所研究的所有噬菌体中，这种失活需要RecA刺激的阻遏物自蛋白分解事件，切割恰好发生在连接这些蛋白两个结构域的“连接物”区域中的丙氨酸-甘氨酸二肽序列中。然而，50 nm，并在37℃下孵育15分钟，以允许形成开放的络合物。加入0.25 mM的ATP、GTP和CTP，0.04 mM的UTP，10(CID：3)Ci的[(Cid：10)-32P]UTP和0.1 mg/ml的肝素，继续孵育10min后，加入甲酰胺染料混合物(90%甲酰胺和1(Cid：5)TbE)停止反应，并在6%变性凝胶上分级。位置和数量的RNA转录本通过荧光成像仪分析(Amersham Biosciences)可视化。RecA和pH刺激的933W抑制子的自切。在标准反应缓冲液(50 mM KCl15 mM Tris[pH 7.5]，2 mM MgCl2，0.1 mM EDTA，2 mM DTT)中，将1.2 5(Cid：3)M RecA(震中)、5 mM(Cid：9)-S-fi(Sigma)和1.5(Cid：3)M寡核苷酸(DT 2 0)(IDT Technologies)混合，在室温下孵育10分钟，形成活化的RecA抑制物。然后，加入20(Cid：3)M933W阻遏剂，在37℃下孵育不同时间，观察20(Cid：3)M933W阻遏剂在含有50 mM 3-(环己氨基)-1-丙磺酸(CAPS)-NaOH(pH 10.3)和50 mM KCl37℃的缓冲液中孵育不同时间对抑制物自切割的影响。
The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O R ). Also typical, when bound at O R , 933W repressor functions as an activator at the P RM promoter and a repressor at the P R promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O L ) contains only two repressor binding sites, but the O L -bound repressor still efficiently represses P L transcription. Lambdoid prophage induction requires inac- tivation of the repressor’s DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a “linker ” region that joins the two domains of these proteins. However, 50 nM, and incubation was continued for 15 min at 37°C to allow the formation of open complexes. The transcription reaction was started by the addition of 0.25 mM ATP, GTP, and CTP, 0.04 mM UTP, 10 (cid:3) Ci of [ (cid:10) - 32 P]UTP, and 0.1 mg of heparin/ml. After 10 min of further incubation, the reactions were stopped by addition of formamide dye mix (90% formamide and 1 (cid:5) TBE) and fractionated on 6% denaturing gels. The positions and amounts RNA transcripts were visualized by Phosphorimager analysis (Amersham Biosciences). RecA- and pH-stimulated autocleavage of 933W repressor. To examine RecA-stimulated autocleavage of 933W repressor, activated RecA filaments were formed by mixing 1.25 (cid:3) M RecA (Epicentre), 5 mM (cid:9) -S-ATP (Sigma), and 1.5 (cid:3) M oligo (dT 20 ) (IDT Technologies) in standard reaction buffer (50 mM KCl, 15 mM Tris [pH 7.5], 2 mM MgCl 2 , 0.1 mM EDTA, 2 mM DTT) and incubating this mixture at room temperature for 10 min. Subsequently, 20 (cid:3) M 933W repressor was added, and this mixture was incubated for various times at 37°C. pH-induced repressor autocleavage was effected by incubating 20 (cid:3) M 933W repressor for various times in a buffer containing 50 mM 3-(cyclohexylamino)-1-propane sulfonic acid (CAPS)–NaOH (pH 10.3) and 50 mM KCl at 37°C. The cleavage reaction was quenched by adding an SDS-containing sample loading buffer, and the reaction products were separated on 15% Tris–Tricine polyacrylamide gels and visualized by staining with Coomassie brilliant blue.