Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L–P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5′ end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.
Eukaryotic transcription requires the assembly of a multi-subunit preinitiation complex (PIC) comprised of RNA polymerase II (Pol II) and the general transcription factors. The co-activator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at sub-4 Å. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
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