Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli.
Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli.
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DOI：
10.1016/s0021-9258(18)46867-6
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发表时间：
1994-11
期刊：
影响因子：
--
通讯作者：
P. Czernik;D. Shin;B. Hurlburt
中科院分区：
文献类型：
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作者：
P. Czernik;D. Shin;B. Hurlburt
大肠杆菌的trp阻遏物通过与受调控启动子内的操纵子序列结合来控制参与色氨酸生物合成的操纵子中的转录起始。天然存在的操纵子在18个碱基对区域上是同源的，并显示二分体对称性。我们已经研究了一个阻遏物结合位点的序列决定因素，使用功能选择/聚合酶链反应（PCR）扩增策略。生成trp阻遏物亲和柱，并用于从合成双链DNA的随机池中选择具有结合能力的DNA。通过加入色氨酸类似物β-吲哚丙烯酸洗脱显示出依赖于色氨酸的高亲和力结合的DNA，并通过PCR扩增。在亲和层析和PCR的迭代循环之后，克隆和测序所选择的DNA。CTAG四核苷酸存在于所有天然操纵子的共有序列中，在所有选定的DNA中均发现。利用阻遏物亲和柱的作图实验表明，CTAG基序是阻遏物结合的关键决定因素。定量电泳迁移率变动分析与纯化的色氨酸阻遏物显示，虽然一些DNA的结合由一个阻遏物二聚体，其他的结合由两个阻遏物二聚体的协同性。测量的结合常数范围为0.035至0.5 nM的选定的DNA，相比之下，0.1 nM的色氨酸操作。
trp repressor of Escherichia coli controls transcription initiation in operons involved in tryptophan biosynthesis by binding to operator sequences within the regulated promoters. Naturally occurring operators are homologous over an 18-base pair region and display dyad symmetry. We have examined the sequence determinants of a repressor binding site using a functional selection/polymerase chain reaction (PCR) amplification strategy. A trp repressor affinity column was generated and used to select binding-competent DNAs from a randomized pool of synthetic double-stranded DNA. DNAs that showed tryptophan-dependent high-affinity binding were eluted by addition of the tryptophan analog beta-indole acrylic acid and amplified by PCR. Following iterative cycles of affinity chromatography and PCR, the selected DNAs were cloned and sequenced. The CTAG tetranucleotide, present in the consensus sequence of all natural operators, was found in all selected DNAs. Mapping experiments utilizing the repressor affinity column showed the CTAG motif to be a critical determinant for repressor binding. Quantitative electrophoretic mobility shift assays with purified trp repressor revealed that although some of the DNAs were bound by one repressor dimer, others were bound by two repressor dimers with cooperativity. Measured binding constants ranged from 0.035 to 0.5 nM for the selected DNAs, compared with 0.1 nM for the trp operator.