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 wild-type 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.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

Cryo-EM structures of human RNA polymerase I

“…7d). Insertion of the DNA-mimicking loop into the cleft could therefore also prevent positioning of the RPA49 linker that assists in closing the clamp in the transition to transcription elongation 28,52 .…”

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

“…Recently, structural analysis of Rpa49CT provided further insights (16,29,41,(57)(58)(59)(60). These results, in combination with genetic data, indicated that the Rpa49 linker together with the Rpa49 tWH domain might stabilize the closed conformation of DNA-bound Pol I, thereby maintaining a narrow cleft (61,62). It is possible that an enzyme containing Rpa49LCT, but no Rpa34.5/Rpa49NT may not enter a locked state with a 3´ RNA displaced from the active center, and, thus, supports high processivity.…”

The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2

“…These results suggest a mechanism by which the surface of A135 (in particular, the ED1) plays a pivotal role in specific factor exchange in Pol I. Recent genetic studies have suggested that A12.2 may be involved in modulation of the movement of the jaw/lobe interface especially in the absence of A49, as the A49 linker and tWH domain appear to stabilize the closed conformation of Pol I when bound to DNA (Darrière et al, 2018). As the A12.2C binds to the A135 ED1, which sits next to the A135 lobe, the A12.2C might restrict the movement of the lobe.…”

“…A12.2N interacts directly with the dimerization domain of A49, thus stabilizing the anchoring of the heterodimer (Engel et al, 2013; Fernández-Tornero et al, 2013). Recently, A12.2 has also been proposed to be important for transcription initiation in vivo and in vitro , especially in the absence of A49 (Darrière et al, 2018). Combined with the reduced number of general transcription factors required for productive transcription initiation, the Pol I A49-A34.5 heterodimer and subunit A12.2 might promote the high initiation rate observed on rDNA repeats (French et al, 2003).…”