VISUALIZATION AND QUANTITATIVE-ANALYSIS OF COMPLEX-FORMATION BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND AN RIBOSOMAL-RNA PROMOTER INVITRO
VISUALIZATION AND QUANTITATIVE-ANALYSIS OF COMPLEX-FORMATION BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND AN RIBOSOMAL-RNA PROMOTER INVITRO
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DOI：
10.1093/nar/16.20.9789
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发表时间：
1988-10-25
影响因子：
14.9
通讯作者：
GOURSE, RL
中科院分区：
文献类型：
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作者：
GOURSE, RL
我们已经在体外建立了稳定RNA聚合酶与rrnB P1启动子相互作用的条件。大肠杆菌启动子对定量复合体形成的要求是不寻常的：(1)需要包含一个竞争对手，以便使特定足迹可视化。(2)由于复杂地层对盐敏感，低盐浓度是必要的。(3)为了防止复合物的解离，需要添加起始核苷酸ATP和CTP，导致二核苷酸的生成速率较低。该复合物已使用dna印迹和过滤器结合试验进行了检测。它的特点是在大多数大肠杆菌启动子中，一个不受DNAase I切割保护的区域略微上游延伸到受RNA聚合酶保护的区域。我们发现每摩尔启动子DNA只需要一摩尔活性RNA聚合酶来检测过滤器结合的复合物。在测量的条件下，RNA聚合酶与rrnB P1的结合速度与报道的其他大肠杆菌或噬菌体启动子的结合速度一样快，甚至更快。
We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.