TRANSCRIPTION OF AN ARTIFICIAL RIBOSOMAL RNA GENE IN THE YEAST SACCHAROMYCES (ENHANCER ELEMENT)

Abstract

Ribosomal RNA is transcribed from a set of 120 tandemly repeated rDNA genes located on chromosome XII in the yeast, Saccharomyces cerevisiae. A genetic analysis of ribosomal precursor RNA synthesis is difficult because reiteration of the rDNA genes precludes the isolation of cis-dominant promoter mutations. To determine what sequences modulate transcription of ribosomal precursor RNA in vivo, a series of artificial rDNA genes containing a fragment of DNA from Eschericia coli bacteriophage T7 were introduced into yeast cells. The inclusion of unique sequences in the artificial genes allows measurement of transcription from a single rDNA repeat despite a background of transcription from genomic rDNA. The artificial genes were introduced into yeast on centromeric plasmids to maintain a constant copy number and select against integration into the genome.;Correct transcription of the artificial gene was observed. Transcripts have the same 5' termini as endogenous 35S rRNA. The size of the transcripts corresponds to that predicted for proper transcription initiation and termination, and they are not polyadenylated. Three regions of ribosomal spacer affect transcription of rRNA. (1) Sequences within 200 bp of the 5' terminus of 35S rRNA support low levels of transcription, but at multiple initiation sites. (2) Sequences more than 200 bp upstream of initiation increase the efficiency of transcription and direct transcription initiation to a single site. (3) Most striking is that a 190 bp region over 2230 bp upstream from the initiation site stimulates transcription 15-fold. This region can direct transcription initiation to a single site. The 190 bp region shares many properties of a eukaryotic enhancer element. It can function in either orientation, upstream or downstream of the apparent transcription initiation site. In addition, it contains 7 bp inverted repeats that are homologous to a consensus sequence found in the SV40 enhancer element. However, it ability to function is strongly influenced by adjacent sequences. Finally, the activity of the 190 bp region is decreased when an rRNA transcription termination sequence is placed between it and the site of initiation, supporting a model in which the 190 bp region serves as an entry site for RNA polymerase I and/or factors that comprise a transcription complex.