Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). ...Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3′ extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5′ end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
Display omitted
•Cryo-EM snapshots of the transcription elongation, termination, and recycling states•After being copied, the template 3′ end rebinds the polymerase in a secondary site•Mechanism of viral mRNA poly(A) tail formation by stuttering elucidated•Efficient reformation of the promoter allows multiple transcripts from one RNP
Influenza polymerase transcribes the negative sense viral RNA genome into mRNA in the nucleus of infected cells. This work by Cusack and colleagues reports high-resolution cryo-EM structures of the polymerase at various stages of transcription providing a molecular basis for the complete transcription cycle, which should enable improved inhibitor design.
Structure and Function of Influenza Polymerase Wandzik, Joanna M; Kouba, Tomas; Cusack, Stephen
Cold Spring Harbor perspectives in medicine,
09/2021, Letnik:
11, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Influenza polymerase (FluPol) plays a key role in the viral infection cycle by transcribing and replicating the viral genome. FluPol is a multifunctional, heterotrimeric enzyme with cap-binding, ...endonuclease, RNA-dependent RNA polymerase and polyadenylation activities. It performs its functions in the context of the viral ribonucleoprotein particle (RNP), wherein the template viral RNA is coated by multiple copies of viral nucleoprotein. Moreover, it interacts with a number of host proteins that are essential cofactors and, consequently, adaptive mutations in the polymerase are required for crossing the avian-human species barrier. In this review, we show how mechanistic understanding of how FluPol performs its multiple functions has greatly advanced over the last decade through determination of high-resolution structures by X-ray crystallography and cryo-electron microscopy. These have revealed not only the detailed architecture of FluPol but highlighted the remarkably conformational flexibility that is inherent to its functioning as a dynamic RNA synthesis machine. Structural studies are also underpinning current attempts to develop next-generation anti-influenza drugs that directly target FluPol.
The endoribonuclease RNase E is a principal factor in RNA turnover and processing that helps to exercise fine control of gene expression in bacteria. While its catalytic activity can be strongly ...influenced by the chemical identity of the 5′ end of RNA substrates, the enzyme can also cleave numerous substrates irrespective of the chemistry of their 5′ ends through a mechanism that has remained largely unexplained. We report structural and functional data illuminating details of both operational modes. Our crystal structure of RNase E in complex with the sRNA RprA reveals a duplex recognition site that saddles an inter-protomer surface to help present substrates for cleavage. Our data also reveal an autoinhibitory pocket that modulates the overall activity of the ribonuclease. Taking these findings together, we propose how RNase E uses versatile modes of RNA recognition to achieve optimal activity and specificity.
Display omitted
•RNase E recognizes RNA secondary structure•Signature on the substrate 5′ end recognizes and activates RNase E•RNase E intrinsic activity is repressed by a conserved autoinhibition motif
RNase E is a central ribonuclease of RNA metabolism and post-transcriptional control of gene expression. Through structural and functional analyses, Bandyra et al. reveal how the enzyme activity can be favored by recognition of the 5′ end and RNA secondary structure. The data also reveal a conserved autoinhibitory motif that suppresses RNase E intrinsic activity.
PDF
