CONSTRUCTION OF BACTERIOPHAGE-T7 LATE PROMOTERS WITH POINT MUTATIONS AND CHARACTERIZATION BY INVITRO TRANSCRIPTION PROPERTIES
CONSTRUCTION OF BACTERIOPHAGE-T7 LATE PROMOTERS WITH POINT MUTATIONS AND CHARACTERIZATION BY INVITRO TRANSCRIPTION PROPERTIES
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DOI：
10.1093/nar/15.13.5413
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发表时间：
1987-07-10
影响因子：
14.9
通讯作者：
BURGESS, RR
中科院分区：
文献类型：
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作者：
CHAPMAN, KA;BURGESS, RR
本文描述了18个克隆的单点突变噬菌体T7晚期启动子的构建，并通过体外转录实验来表征这些启动子的性质。由于突变的启动子被克隆到相同的背景中，在转录测定中观察到的差异直接归因于点突变。所有突变的启动子都比野生型活性低，但它们可以分为两种类型。A型突变从-4映射到+1，并且当模板线性化或当60 mM NaCl加入到反应缓冲液中时降低启动子活性。B型突变从-9映射到-7，并且在所有测试条件下降低启动子活性。在几个位点，所有三种可能的点突变都是可用的。在这些网站上，我们观察到的层次结构的碱基对的偏好，由启动子活性，这可能表明T7 RNA聚合酶与大沟中的基团相互作用。
This paper describes the construction of 18 cloned bacteriophage T7 late promoters with single point mutations.In vitrotranscription experiments were used to characterize the properties of these promoters. Since the mutated promoters are cloned into identical backgrounds, differences seen in the transcription assays are directly attributable to the point mutations.All of the mutated promoters are less active than wildtype, but they can be divided into two types. Type A mutations map from −4 to +1 and reduce promoter activity when the template is linearized or when 60mM NaCl is added to the reaction buffer. Type B mutations map from −9 to −7 and reduce promoter activity under all conditions tested. At several sites all three possible point mutations are available. At these sites we observed hierarchies of base pair preference, as determined by promoter activity, that may indicate that T7 RNA polymerase interacts with groups in the major groove.