Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes

Abstract

Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide. Two of these regions have been molecularly cloned. Each contains a sequence related not only to the D. melanogaster DNA fragment that was used as a probe in its isolation but also to the immediately adjacent DNA fragment of the D. melanogaster RNA polymerase II gene. The two cloned S. cerevisiae DNA sequences are each the template for single transcripts in vivo, one of 5.9 kilobases and the other of 4.6 kilobases. In vitro translation of hybrid-selected cellular RNA indicated that the former locus encodes a protein of Mr 220,000, equal in size to the largest polypeptide subunit of S. cerevisiae RNA polymerase II. Disruption of either gene by targeted integration of URA3+ DNA demonstrated that each is single-copy and essential in a haploid genome. We suggest that these S. cerevisiae loci are members of a family of related genes encoding the largest subunit polypeptides of RNA polymerases I, II, and III.