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首页> 外文期刊>Journal of bacteriology >Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector
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Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector

机译：利用新开发的启动子克隆载体对平稳相启动子进行分类和强度测定

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When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.
机译：当大肠杆菌培养物从指数生长转变为固定相时，与生长相关的基因的表达趋于平稳，而许多固定相特异的基因被打开。为了深入了解基因组转录的生长阶段依赖性全局调控，我们分析了与固定相基因相关的启动子的强度和特异性。为了对启动子活性进行体内分析，分离了翻译起始密码子上游的300至500 bp DNA片段，并将其插入到新构建的双荧光蛋白（DFP）载体中。通过测量绿色荧光蛋白（GFP）来确定测试启动子的活性。为避免质粒拷贝数的可能影响，通过测量红色荧光蛋白（RFP）来确定参考启动子 lac UV5在同一质粒上的转录水平。因此，通过确定GFP / RFP比可以容易而准确地确定测试启动子的活性。对100个测试启动子的培养时间依赖性变异的分析表明：（i）主要的固定相启动子组仅在RpoSσ存在时才被上调; （ii）即使在没有RpoS的情况下，某些启动子的活性也会发生相耦合的增加; （iii）在没有RpoS的情况下某些启动子的活性增加。通过使用重组的RpoD和RpoS全酶测试体外转录，证实了该分类。

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