RRN3 GENE OF SACCHAROMYCES-CEREVISIAE ENCODES AN ESSENTIAL RNA-POLYMERASE-I TRANSCRIPTION FACTOR WHICH INTERACTS WITH THE POLYMERASE INDEPENDENTLY OF DNA-TEMPLATE

RRN3 is one of the RRN genes specifically required for the transcripti on of rDNA by RNA polymerase I (Pol I) in Saccharomyces cerevisiae, We have cloned the gene, determined the nucleotide sequence, and found t hat it is an essential gene which encodes a protein of calculated mole cular weight of 72 369. Extracts prepared from rrn3 mutants were defec tive in in vitro transcription of rDNA templates. We used extracts fro m a strain containing an epitope-tagged Rrn3 protein to purify a facto r that could complement the mutant extracts. Using immunoaffinity puri fication combined with Mono Q chromatography, we obtained an essential ly pure preparation of Rrn3p which complements the mutant extracts. By carrying out template commitment experiments, we found that Rrn3p is not part of the pre-initiation complex that is stable through multiple rounds of transcription. We also found that pre-incubation of Rrn3p w ith purified Pol I leads to stimulation of transcription upon subseque nt mixing with DNA template and other transcription reaction component s. Single-round transcription experiments using the detergent Sarkosyl showed that this stimulation is due to increased efficiency of format ion of a Sarkosyl-resistant pre-initiation complex. Thus, Rrn3p appear s to interact directly with Pol I, apparently stimulating Pol I recrui tment to the promoter, and is distinct from two other pol I-specific t ranscription factors, the Rrn6/7 complex and the Rm5/9/10 complex (UAF ), characterized previously.