Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae

Abstract

Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3′ end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from −209 bp.