Structural features of the DNA template required for transcription in vitro by yeast RNA polymerase B (II).
Yeast RNA polymerase II initiates in vitro transcription at two sites located within the vector DNA and the cloned promoter, on a recombinant plasmid DNA containing the yeast iso1 cytochrome c promoter. Both initiation sites are found within a DNA fragment hypersensitive to osmium tetroxide modification. Using a series of yeast iso1 cytochrome c promoter deletions, we have characterized an upstream DNA sequence required for optimal transcription from this site and shown in this case a correlation between osmium sensitivity and the capacity of RNA polymerase to initiate. However, perturbation of the double helix is not sufficient to generate a transcription initiation site. Insertion of 28 alternating AT residues at the EcoRV site of pBR322 generates an site hypersensitive to osmium tetroxide modification, that does not serve as a transcription start site.
D001665:Binding Sites; D004271:DNA, Fungal / Q000378:metabolism*; D004274:DNA, Recombinant; D009993:Osmium Tetroxide; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004274:DNA, Recombinant; D012319:RNA Polymerase II; D009993:Osmium Tetroxide