An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled.
Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.
Leblanc
B P
BP
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 6, Room B1-12, Bethesda, MD 20892-2715, USA.
Benham
C J
CJ
Clark
D J
DJ
D002352:Carrier Proteins; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008668:Metallothionein / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C007121:copper thionein; D008668:Metallothionein; D012319:RNA Polymerase II