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 transcription of rDNA by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. We have cloned the gene, determined the nucleotide sequence, and found that it is an essential gene which encodes a protein of calculated molecular weight of 72 369. Extracts prepared from rrn3 mutants were defective in in vitro transcription of rDNA templates. We used extracts from a strain containing an epitope-tagged Rrn3 protein to purify a factor that could complement the mutant extracts. Using immunoaffinity purification combined with Mono Q chromatography, we obtained an essentially 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 with purified Pol I leads to stimulation of transcription upon subsequent mixing with DNA template and other transcription reaction components. Single-round transcription experiments using the detergent Sarkosyl showed that this stimulation is due to increased efficiency of formation of a Sarkosyl-resistant pre-initiation complex. Thus, Rrn3p appears to interact directly with Pol I, apparently stimulating Pol I recruitment to the promoter, and is distinct from two other Pol I-specific transcription factors, the Rrn6/7 complex and the Rrn5/9/10 complex (UAF), characterized previously.
Yamamoto
R T
RT
Department of Biological Chemistry, University of California, Irvine, CA 92717, USA.
Nogi
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Y
Dodd
J A
JA
Nomura
M
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000378:metabolism; D000939:Epitopes; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D035463:Pol1 Transcription Initiation Complex Proteins; D012318:RNA Polymerase I / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D000939:Epitopes; D005656:Fungal Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; C101161:RRN3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012318:RNA Polymerase I