Selective transcription of hybrid plasmids containing yeast rDNA was achieved with a template-dependent S100 fraction from whole cell extracts of Saccharomyces cerevisiae. A small region of the yeast rDNA which directs selective initiation in vitro was identified by subcloning. An initiation site was mapped within this region on the basis of the molecular weights of transcripts synthesized in vitro from templates which were cleaved with restriction endonucleases at a series of sites downstream from the site of initiation. The initiation site maps to a position 3.0 kilobase pairs upstream from the sequences which encode the 5' terminus of 18 S rRNA. In vitro initiation from this site is not inhibited by 50 micrograms/ml of alpha-amanitin and is completely abolished when the reactions contain 0.2 M (NH4)2SO4. Based on these data, selective transcription of yeast rDNA in vitro is RNA polymerase I-dependent. Several S1 nuclease-resistant hybrids are formed between DNA probes labeled at restriction endonuclease sites downstream from the in vitro initiation site and high molecular weight cellular RNA. The 5' terminus of the most abundant rRNA precursor maps approximately 0.7 kilobase pair upstream from sequences which encode the 5' terminus of 18 S rRNA. This corresponds to the 5' terminus of the 35 S rRNA precursor reported by others. The 5' terminus of the largest detectable precursor synthesized in vivo corresponds closely with the initiation site identified in vitro. Based on the data presented here, RNA polymerase I traverses the interspersed 5 S rRNA gene. Since these two ribosomal genes are transcribed in opposite directions, this arrangement of the RNA polymerase I and III promoters may ensure that equivalent amounts of the two gene products are synthesized in vivo.

• PMID: 6298229
• PubMed

Reference Type

Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Swanson ME, Holland MJ

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