In vitro transcription of the yeast alcohol dehydrogenase I gene by homologous RNA polymerase B (II). Selective initiation and discontinuous elongation on a supercoiled template.

A new in vitro approach is used to investigate the specificity of purified yeast RNA polymerase B (II). The template is supercoiled, the transcription is primed by a dinucleotide, and the transcripts are analyzed by polyacrylamide gel electrophoresis after synthesis in the absence of one nucleoside triphosphate. Under these conditions, two recombinant plasmids carrying the gene or part of the gene for yeast alcohol dehydrogenase I direct the synthesis of a very limited number of oligonucleotides. Elongation of these prelabeled oligomers, using unlabeled substrates, occurs in a discontinuous way. A major transcript of 200 nucleotides accumulates transiently. Southern hybridization shows that it is initiated about 1,400 bases upstream from the origin of the yeast alcohol dehydrogenase I gene. A minor start was identified, by a modified runoff experiment, at position -35 from the AUG initiation codon. The location of this site is related to the presumptive in vivo transcription starts. The selectivity disappears when the template is a truncated DNA. Then, initiation occurs predominantly at nicks introduced by the restriction enzymes.