The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3.
RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA). The mechanism of Pol I recruitment onto rDNA promoters is poorly understood. Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I-Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality. Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I-Rrn3 complex. Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography. Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor. The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes.
Peyroche
G
G
Service de Biochimie et de Génétique Moléculaire, CEA/Saclay, F-91191 Gif sur Yvette Cedex, France.
Milkereit
P
P
Bischler
N
N
Tschochner
H
H
Schultz
P
P
Sentenac
A
A
Carles
C
C
Riva
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D007091:Image Processing, Computer-Assisted; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D035463:Pol1 Transcription Initiation Complex Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021122:Protein Subunits; D012318:RNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D005656:Fungal Proteins; D046911:Macromolecular Substances; D035463:Pol1 Transcription Initiation Complex Proteins; D021122:Protein Subunits; C101161:RRN3 protein, S cerevisiae; C090311:RRN6 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012318:RNA Polymerase I