Characterization of an RNA polymerase I-dependent promoter within the spacer region of yeast ribosomal cistrons.

Nucleotide sequences which are required for RNA polymerase I-dependent selective initiation of transcription in vitro from a site within the spacer region of cloned yeast ribosomal DNA have been identified. Yeast rDNA templates containing deletion mutations extending from restriction endonuclease cleavage sites located upstream and downstream from the transcriptional initiation site were constructed. The ability of these mutant templates to support selective transcription in vitro was determined using a yeast whole cell extract. Nucleotide sequences which are required for selective transcription in vitro are within a 22-base pair region which is located immediately adjacent to the transcriptional initiation site. The 3' boundary of this 22-base pair sequence was mapped within a single base pair and resides within the transcribed portion of the rDNA. Nucleotide sequences upstream and downstream from the 22-base pair region are not required for selective transcription and do not appear to affect the efficiency of transcription in vitro. A hybrid plasmid containing only 32 base pairs of yeast rDNA, which includes the 22-base pair region, supports efficient and accurate RNA polymerase I-dependent transcription in vitro. These data demonstrate that the 22-base pair region of yeast rDNA is sufficient for accurate initiation of transcription in vitro. The transcriptional properties of several cloned rDNA templates isolated from two haploid yeast strains and a strain of bakers' yeast were examined. Four cistrons were identified which differ in nucleotide sequence. Three cistrons contain the 22-base pair promoter region and they support selective transcription in vitro. The fourth cistron does not support selective transcription in vitro and contains a single base pair substitution within the 22-base pair promoter sequence.