Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing.
Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing.
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大肠杆菌启动子杂交体和改变-35至-10间距的启动子突变体的体内活性的构建和分析。
DOI：
10.1016/0378-1119(82)90042-7
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发表时间：
1982
期刊：
影响因子：
3.5
通讯作者：
Bennett,GN
中科院分区：
文献类型：
--
作者：
Russell,DR;Bennett,GN
通过交换flacuv5、tet和trp启动子的−35和−10区域，构建了一系列启动子杂合体。这三个启动子和由它们构建的六个杂交启动子被插入到pKO质粒中，该质粒将半乳糖激酶的表达置于插入的启动子控制之下。此外，制备的启动子突变体改变了启动子- 35和- 10区域之间的间距。在这个间隔区有一个或两个额外碱基对的四联体启动子衍生物和两个不同间隔的ac:：四联体启动子的构建被插入到半乳糖激酶表达质粒中。在含有这些质粒的菌株中测量半乳糖激酶水平，可以比较启动子的体内活性。最强的杂化启动子（阶：−35，−10）为trp::lacandtrp:: te3，表明trp启动子的−35区效率较高。最弱的启动子是：：trp，lac::trpandlac:: tete，这表明trppromoter存在一个弱的- 10区域，以及- 35到- 10间距的重要性。对间隔差异相关启动子的活性分析表明，距离为19 bp的启动子活性非常弱，18 bp的启动子活性低于17 bp的启动子活性，17 bp是启动子中最常见的间隔。
A series of promoter hybrids has been constructed by exchanging the − 35 and − 10 regions oflacUV5,tet, andtrppromoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the − 35 and − 10 regions of the promoter. Derivatives of thetetpromoter with one or two extra base pairs in this spacer region and constructions of thelac::tethybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: − 35, − 10) weretrp::lacandtrp::tetsuggesting a high efficiency for the − 35 region of thetrppromoter. The weakest promoters weretet::trp,lac::trpandlac::tetindicating a weak − 10 region for thetrppromoter and the importance of − 35 to − 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.