Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Abstract

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo , the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro . We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.

Conserved strategies of RNA polymerase I hibernation and activation

“…Helix α0 forms only in Sc Pol I and was presumed to strengthen stalk attachment compared to Pol II 5 , 6 . A divergent region (residues 549–559) and an insertion (residues 591–600) in the ‘external 2’ domain of Sp subunit A135 are adjacent to the lobe-region responsible for tight association of the A49/34.5 sub-complex 18 and may be responsible for its reduced affinity (compare Supplementary Figs. 3b and 4 ).…”

The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same

“…The second question is significant, because studies in yeast suggest that while the heterodimer may play significant roles in rDNA transcription, it is not essential [ 21 , 24 , 45 , 46 , 50 , 63 ]. However, genetic screens of the human genome have suggested that both proteins are “essential” for mammalian cell proliferation [ 64 , 65 , 66 ].…”

Nucleolar localization of the yeast RNA exosome subunit Rrp44 hints at early pre-rRNA processing as its main function

“…Exosome is also involved in quality control steps of rRNA processing, targeting 23S rRNA generated by direct cleavage at the A3 site for degradation, and unprocessed 35 S rRNA (Fig. S1B) (20)(21)(22). Interestingly, despite the direct involvement of the exosome in 7S pre-rRNA processing and quality control, 60S subunits bearing 7S have been identified in polysomes of exosome cofactors mutants (23).…”