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RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template.
Author(s) -
Yamamoto R. T.,
Nogi Y.,
Dodd J. A.,
Nomura M.
Publication year - 1996
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1996.tb00770.x
Subject(s) - biology , genetics , polymerase , rna polymerase ii , transcription factor ii d , rna polymerase i , dna polymerase , saccharomyces cerevisiae , rna polymerase ii holoenzyme , rna polymerase , transcription (linguistics) , gene , microbiology and biotechnology , rna dependent rna polymerase , rna , promoter , gene expression , linguistics , philosophy
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.