Pure yeast RNA polymerase B (II) initiates transcription at specific points on supercoiled yeast DNA

Pure yeast RNA polmymerase B (II) can selectively initiate abortive transcription on a supercoiled recombinant plasmid DNA carrying yeast DNA in the presence of low concentrations of ribonucleoside triphosphates and Mn2+. Five major products ranging between 60 and 150 nucleotides were characterized by hybridization. Three of them originate from the vector pBR322 and two from the yeast DNA insert. Based on a RNA primer extension reaction with recombinant M13 DNAs as template, a method allowing the mapping of the short abortive RNA products has been developed. An initiation site within the yeast DNA insert has thus precisely been mapped. The DNA sequence in this region was determined and showed several relevant features. The in vitro initiation site is preceded by a potential TATATATA box at -40 base pairs and at -105 by the sequence GTTAATCT similar to the consensus sequence GCTCAATCT usually found around 80 base pairs upstream from the cap site. Large blocks of alternated purine pyrimidine residues are found in this region as for several known yeast promotors. The 5' end of the RNA initiated from this site contains several potential signals for the initiation of translation. The possibility that a B to Z transition of DNA could be important for the interaction of the RNA polymerase with its template is discussed.