Butlin M, Quincey R
School of Biochemistry, University of Birmingham, Edgbaston, U.K.
Yeast. 1991 Oct;7(7):679-89. doi: 10.1002/yea.320070703.
The promoter and enhancer of the rRNA gene of Saccharomyces cerevisiae have been studied using a nuclease S1 protection assay to detect transcripts of an rRNA minigene in transformed yeast. Analysis of 5' deletion mutants showed that DNA between -163 bp and -155 bp was important for promoter activity and that some DNA between -155 bp and -145 bp was essential. The importance of DNA far upstream from the initiation site was confirmed by showing that minigene expression was much reduced by linker scanner mutations clustered around -148 bp, -133 bp and -100 bp, and was abolished by mutations clustered around -118 bp. The enhancer for rRNA biosynthesis increased transcription from all of the five mutated promoters that were tested. The magnitude of the enhancer effects on weakly active promoters was two- to three-fold less than on the wild-type promoter. Expression of a minor transcript in a 5' deletion to -10 bp was substantially reduced by a mutation which altered two base pairs in the core sequence of the promoter-proximal REB1 binding site.
利用核酸酶S1保护分析法检测转化酵母中rRNA小基因的转录本，对酿酒酵母rRNA基因的启动子和增强子进行了研究。对5'缺失突变体的分析表明，-163 bp至-155 bp之间的DNA对启动子活性很重要，-155 bp至-145 bp之间的一些DNA是必不可少的。通过显示小基因表达因聚集在-148 bp、-133 bp和-100 bp附近的接头扫描突变而大幅降低，并因聚集在-118 bp附近的突变而被消除，证实了起始位点上游很远的DNA的重要性。rRNA生物合成的增强子增加了所测试的五个突变启动子的转录。增强子对弱活性启动子的作用程度比对野生型启动子小两到三倍。一个5'缺失至-10 bp的次要转录本的表达因一个突变而大幅降低，该突变改变了启动子近端REB1结合位点核心序列中的两个碱基对。
