Activity of promoter mutants of the yeast ribosomal RNA gene with and without the enhancer.
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.
Butlin
M
M
School of Biochemistry, University of Birmingham, Edgbaston, U.K.
Quincey
R
R
D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005656:Fungal Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C065681:REB1 protein, S cerevisiae; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015719:Single-Strand Specific DNA and RNA Endonucleases