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.

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