Highlights d Crystallographic structure of the human eIF4A1,AMPPNP,RocA,polypurine RNA complex d Direct base recognition by RocA induces polypurine RNA selectivity on eIF4A1 d Natural amino acid substitutions found in Aglaia eIF4As provide self-resistance to RocA

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Purification of human eIF2The α-, β-, and γ-subunits of human eIF2, and the eIF2-specific chaperone protein human Cdc123 (33) were co-expressed in FreeStyle 293-F cells, using the four pEBMulti-Neo plasmid vectors (Wako), and eIF2γ was expressed with C-terminal FLAG and His8 tags. The cells were lysed in buffer A [20 mM MES-KOH buffer (pH 6.0), containing 150 mM KCl, 1 mM MgCl2, 10%(v/v) glycerol, and 5 mM 2-mercaptoethanol] supplemented with 20 mM imidazole, 0.5 mM EDTA, 0.1%(v/v) Triton X-100 and protease inhibitors. After 30 min on ice and centrifugation, the supernatant was applied to a HisTrap (GE Healthcare) column equilibrated with buffer A supplemented with 20 mM imidazole, and eluted with a linear gradient of 20-500 mM imidazole. The fraction containing eIF2 was collected and applied to a HiTrap SP (GE Healthcare) column equilibrated with buffer A, and eluted with a linear gradient of 200-640 mM KCl. After three-fold dilution with buffer B [20 mM HEPES-KOH buffer (pH 7.5) containing 100 mM KCl, 0.1 mM MgCl2, 10%(v/v) glycerol, and 1 mM DTT], the sample was applied to a HiTrap Heparin (GE Healthcare) column equilibrated with buffer B, and eluted with a linear gradient of 0.2-1 M KCl. The fraction containing eIF2 was further purified on a Superdex200 (GE Healthcare) column equilibrated with buffer B. eIF2 was phosphorylated by PKR, as described for Komagataella pastoris eIF2 (14). Purification of human eIF2BHuman eIF2Bα and eIF2Bβγδε were purified separately.The fragment encoding human eIF2Bα was cloned into pET-28c (Novagen), in which the thrombin cleavage site was replaced by the HRV 3C protease cleavage site. The T7 Express

Summary The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro , eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin–dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin–dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin–dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Highlights d Cryo-EM structures of the HCV IRES in complex with the translating 80S ribosome d Single-molecule experiments revealed that the HCV IRES binds to the active ribosome d The HCV IRES binds to both the cap-and HCV IRESdependently initiated ribosomes d Concurrent cap-dependent translation enhances HCV IRESdependent translation