Molecular insight into RNA polymerase I promoter recognition and promoter melting

Sadian, Yashar; Baudin, Florence; Tafur, Lucas; Murciano, Brice; Wetzel, René; Weis, Félix; Müller, Christoph W.

doi:10.1038/s41467-019-13510-w

Cited by 40 publications

(60 citation statements)

References 47 publications

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“…The WH3 and WH4 domains are not visible in the cryo-EM reconstructions, indicating that they are mobile and suggesting a function in transcription initiation. Similar functions are reported for the WH domains of yeast Pol I (A49 subunit) 32 , human Pol II (RAP30 subunit of human TFIIF) 30 and yeast Pol III (C34 subunit) [16][17][18] . The majority of residues in RPC5 are not predicted to bind DNA (Extended Data Fig.…”

Section: Extended Structure Of Human Rpc4-rpc5supporting

confidence: 77%

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Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

Girbig¹,

Misiaszek²,

Vorländer³

et al. 2020

Preprint

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ABSTRACTRNA polymerase III (Pol III) synthesises tRNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumour transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Pol III inhibition increases longevity in different animals but also promotes intracellular bacterial growth owing to its role in the immune system. This highlights the importance to better understand human Pol III transcription on a molecular level. Here, we present cryo-EM structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III purified from human suspension cells that were gene-edited by CRISPR-Cas9. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures also resolve elements absent from S. cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron-sulphur cluster in RPC6, which tethers the heterotrimer subcomplex to the Pol III core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by the tumour suppressor MAF1, which may explain why overexpressed RPC7α enhances tumour transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and might contribute to developing inhibitors that selectively target Pol III for therapeutic interventions.

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Section: Extended Structure Of Human Rpc4-rpc5supporting

confidence: 77%

“…8c). The visible part of the N-terminal extension (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46) overlaps with the position of the RPC6 FeS domain (Extended Data Fig. 8d).…”

Section: The Fes Domain Ties Heterotrimer and Corementioning

confidence: 99%

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

Girbig¹,

Misiaszek²,

Vorländer³

et al. 2020

Preprint

Self Cite

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“…During the final stages of revision of this work, a related study was published 72 . Sadian et al provide an excellent description of CF-promoter contacts in detail and investigate the role of an acidic loop in Rrn3, based on higher resolution reconstructions.…”

Section: Discussionmentioning

confidence: 99%

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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“…The tWHD1 is formed by two juxtaposed winged helix domains that form a compact globular domain with one of the two recognition helices, typically involved in DNA binding, buried within the structure ( Figure 3b). The compact conformation of tWHD1 is observed also in solution as highlighted by small-angle x-ray scattering (SAXS) data (Figure 3d, Extended Data Comparison with existing protein structures using the Dali server (http://ekhidna.biocenter.helsinki.fi/dali_server/) suggested similarities between tWHD1 and the WHD of S. cerevisiae Pol II general transcription factor TFIIF Rap30 subunit [24][25][26] and with the tWHD of Pol I A49 subunit [27][28][29] . Both subunits are orthologs of RPC5 and involved in stabilization of the pre-initiation complexes (PICs), suggesting a putative functional link.…”

Section: Structure Of the Rpc5 C-terminal Extensionmentioning

confidence: 89%

Structure of human RNA Polymerase III

Ramsay

Abascal-Palacios

Daiß

et al. 2020

Preprint

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ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.

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Molecular insight into RNA polymerase I promoter recognition and promoter melting

Cited by 40 publications

References 47 publications

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

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

Structure of human RNA Polymerase III

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