RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template.

Abstract

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.

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

“…When detached from DNA, free monomeric Pol I cannot directly be recruited to rDNA promoters but requires prior binding to the activating factor Rrn3 [12,13]. This event allows direct downregulation of rRNA production by Rrn3 degradation [24,30].…”

“…While the CF is sufficient for promoter recognition, UAF and TBP enhance rDNA transcription [10]. Promoter-attached initiation factors foster binding of RNA polymerase I (Pol I) in complex with the Rrn3 protein [11–13]. The complete assembly of enzyme and factors on the promoter constitutes the pre-initiation complex (PIC) that has been proposed to evolve from a closed to an open complex upon DNA melting, which in Pol I is independent from ATP hydrolysis [14–16].…”

Multiple interactions between RNA polymerase I, TIF‐IA and TAF I subunits regulate preinitiation complex assembly at the ribosomal gene promoter

“…Extracts prepared from serum-starved or cycloheximide-treated cells lack this activity and are therefore transcriptionally inactive. TIF-IA corresponds to factor C* (Brun et al, 1994) and yeast Rrn3p (Yamamoto et al, 1996). Both in yeast and mammals, Rrn3p/TIF-IA associates with a subpopulation of Pol I to form the transcriptionally active enzyme, defined as the Pol I entity that is capable of initiating transcription from the rDNA promoter.…”

Identification of factors that promote biogenesis of tRNACGASer

“…RPA49 encodes a nonessential subunit of RNA polymerase I (Pol I) [34] whereas RRN3 codes for an essential Pol I transcription factor [35]. The MOT1 gene codes for an essential protein that has global effects on gene expression by controlling the distribution and activity of the TATA-binding protein (TBP) at promoters of RNA polymerase II (Pol II) transcribed genes [36–40].…”

“…The Mot1 protein is thought to influence both Pol I and Pol II transcription [36–41,46] whereas the function of Rpa49 and Rrn3 appears to be restricted to Pol I transcription [34,35,42]. The mechanisms by which Mot1, Rpa49 and Rrn3 influence tRNA biogenesis is unclear, but it is possible that they indirectly influence the process through their involvement in Pol I transcription.…”