Cryogenic-Electron Microscopy Structures of Viral and Eukaryotic Transcription Complexes

Abdella, Ryan

doi:https://doi.org/10.21985/n2-rxka-cb75

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Cryogenic-Electron Microscopy Structures of Viral and Eukaryotic Transcription Complexes

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Transcription of nucleic acids (DNA and RNA) is a hallmark of life, taking the information stored within genomic nucleic acids and converting it into a form that is useful for producing the proteins necessary for cellular and organismal function. In eukaryotes, transcription of DNA into messenger RNA (mRNA) requires the formation of a 56-subunit pre-initiation complex comprised of RNA Polymerase II (Pol II), the co-activator Mediator, and a group of general transcription factors. Mediator facilitates the assembly of this complex at gene promoters and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by CDK7, a subunit of the cyclin-activated kinase (CAK) module of TFIIH. To understand the mechanism of this process, I have used cryo-electron microscopy to solve the structure of the human Mediator-bound PIC to 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, suggesting the former helps position the latter.Paramyxoviruses are enveloped, non-segmented, negative-strand (NNS) RNA viruses that cause a broad 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 mostly unknown due to the lack of high-resolution structures of this complex in this viral family. In this study, I determined the structure of the L-P complex from parainfluenza virus 5 (PIV5) at 4.3 Å resolution using cryo-electron microscopy. P-OD binds to the RNA-dependent RNA polymerase (RdRp) domain of L and protrudes away from it, while the X domain (XD) 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 novel conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase (PRNTase) 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.

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