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

Tafur, Lucas; Sadian, Yashar; Hanske, Jonas; Wetzel, René; Weis, Félix; Müller, Christoph W.

doi:10.7554/elife.43204

Cited by 39 publications

(38 citation statements)

References 63 publications

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“…The Cterminal domain of subunit A12.2 shows only residual density in funnel domain of subunit A190 ( Supplementary Fig. 2b), but is not localized on the A135 lobe as observed in a 12-subunit EC 43 . Our eiPIC reconstruction shows strong density for the A49/A34.5 dimerization and A34.5 C-terminal tail domains ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 80%

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

Pilsl

Engel

2020

Nat Commun

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Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the 'core factor' subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.

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Section: Resultsmentioning

confidence: 80%

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

Pilsl

Engel

2020

Nat Commun

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“…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.…”

Section: Discussionmentioning

confidence: 97%

“…Accordingly, a dynamic position of Rpa12.2CT in the funnel of subunit Rpa190 depends on the transcriptional status of the elongating Pol I and might be a prerequisite to overcome transcriptional barriers (19,20,26,29,29,41,57,60,71,72). Furthermore, it was suggested that competition between Rpa12.2CT and the heterodimer Rpa34.5/Rpa49 for binding of the Pol I lobe might result in the reversible binding of the heterodimer which could contribute to modulate Pol I activity (62). Such a dynamic interaction could also influence movement through nucleosomes.…”

Section: Discussionmentioning

confidence: 99%

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

Merkl¹,

Pilsl²,

Fremter

et al. 2020

Journal of Biological Chemistry

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RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

RNA polymerase I passage through nucleosomes depends on its lobe binding subunits

Tschochner

Merkl

Pilsl

et al. 2019

Preprint

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RNA polymerase I (Pol I) is a highly efficient enzyme specialized to synthesize most of the ribosomal RNA. After nucleosome deposition at each round of replication the Pol I transcription machinery has to deal with nucleosomal barriers. It was suggested that Pol I-associated factors facilitate chromatin transcription, but it is not known whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here we used in vitro transcription assays to study purified Pol I of the yeast S. cerevisiae and Pol I mutants in comparison to Pol II and Pol III to pass a nucleosome. Under identical conditions, purified Pol I and Pol III, but not Pol II, were able to transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. The contribution of Pol I specific subunit domains to efficient passage through nucleosomes in context with transcription rate and processivity is discussed.

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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

Cited by 39 publications

References 63 publications

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

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

RNA polymerase I passage through nucleosomes depends on its lobe binding subunits

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