The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3

Abstract

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 coexpressed 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 identi®ed the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by af®nity 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.

RNA polymerase I structure and transcription regulation

“…Third, A43 phosphorylation apparently counteracts Pol I dimerization and promotes Rrn3 binding, because (1) A43 binds Rrn3 (ref. 29) and is phosphorylated in Pol I-Rrn3 complexes 39 , (2) A43 phosphorylation sites 40 at Ser 208, Ser 220, Ser 262/263 and Ser 285 are exposed in our structure and face Rrn3 (ref. 34), and (3) Pol I dephosphorylation prevents Rrn3 binding and results in low transcription activity 39 , and extracts from non-growing cells lack active Pol-IRrn3 complexes 38 .…”

“…The defined structure of the connector and its specific interactions with the polymerase cleft argue that connector-mediated polymerase dimerization is functionally significant. Consistent with this, the connector cannot be accommodated in a RESEARCH ARTICLE modelled contracted state of Pol I, and a C-terminal truncation of A43 that deletes the connector results in thermosensitivity in vivo 29 , and is lethal when combined with other Pol I mutations 33 .…”

“…1b), explaining the known interaction between A43 and Rpb6 (ref. 29), and loss of A43 and Rpb6 from Pol I after A14 deletion 32 . Stalk-mediated Pol I dimerization was also observed by electron microscopy 6 , suggesting that the dimer observed in the crystal corresponds to that formed in solution.…”

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

“…In Saccharomyces cerevisiae, Rrn3p has been found to interact with RPA43, a unique subunit of Pol I (Peyroche et al, 2000;Fath et al, 2001). Given that functionally important protein-protein interactions are evolutionarily conserved, we examined whether the homologous mammalian subunit mediates the interaction with TIF-IA.…”

Breaking the mold: structures of the RNA polymerase I transcription complex reveal a new path for initiation

“…The Rrn6 C-terminus, the majority of which is not resolved in the CF crystal structure, is necessary for Rrn3 interaction in yeast two-hybrid studies [3,12]. A striking feature of the Pol I PIC structures is the distance between CF and Rrn3.…”