Cryo-EM uncovers a sequential mechanism for RNA polymerase I pausing and stalling at abasic DNA lesions

Author:

Santos-Aledo AliciaORCID,Plaza-Pegueroles AdriánORCID,Sanz-Murillo MartaORCID,Ruiz Federico M.ORCID,Xu Jun,Gil-Carton DavidORCID,Wang DongORCID,Fernández-Tornero CarlosORCID

Abstract

AbstractRNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites can slow nucleotide addition by base sandwiching between the RNA 3’-end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe mechanism to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.

Publisher

Cold Spring Harbor Laboratory

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