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 0 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 0 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. ll

Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic vRNA into mRNA. The polymerase is initially bound to a promoter comprising the partially base-paired 3′ and 5′ extremities of the vRNA. A short, capped primer, ′cap-snatched′ from a nascent host polymerase II transcript, is directed towards the polymerase active site to initiate RNA synthesis. Here we present structural snapshots, determined by X-ray crystallography and cryo-electron microscopy, of actively initiating influenza polymerase as it transitions towards processive elongation. Unexpected conformational changes unblock the active site cavity to allow establishment of a nine base-pair template-product RNA duplex before the strands separate into distinct exit channels. Concomitantly, as the template translocates, the promoter base-pairs are broken and the template entry region is remodelled. These structures reveal new details of the influenza polymerase active site that will help optimize nucleoside analogs or other compounds that directly inhibit viral RNA synthesis.

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.The initiation phase of protein synthesis is promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The multiprotein eIF3 complex, together with eIFs 1, 1A, and 5, promotes recruitment of the MettRNA i Met /eIF2/GTP ternary complex (TC) to the small ribosomal subunit (40S), producing the 43S preinitiation complex (PIC). At least in yeast, eIFs 1, 3, and 5 and the TC occur in a preformed unit called the multifactor complex (MFC), which enhances the efficiency of the 43S PIC assembly process (reviewed in reference 20). The eIF4F complex, containing the cap-binding eIF4E and the scaffold protein eIF4G, then mediates recruitment of an mRNA to the 43S PIC with the help of eIF3 and the poly(A)-binding protein. The resulting 48S PIC traverses the 5Ј untranslated region (UTR) of mRNA, searching usually for the first AUG codon while unwinding secondary structures in an ATP-dependent reaction stimulated by helicases eIF4A and eIF4B (reviewed in reference 39). This intricate process is called scanning, and its precise molecular mechanism is still poorly understood. It is known that the presence of the TC and eIFs 1, 1A, and 3 in reconstituted mammalian 43S PICs is sufficient for scanning through the unstructured leaders of model mRNAs (38). eIFs 1 and 1A are thought to promote scanning by induction of a conformational change of the 40S head. This change, characterized by opening the latch formed by helices 18 (h18) and 34 (h34) of 18S rRNA and establishing a new interaction between ...

Electron cryomicroscopy suggests regulation of ATM by a dynamic equilibrium between open and closed dimers.