ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

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).

The NF-κB regulator IκBβ exhibits different molecular interactivity and phosphorylation status from IκBα in an IKK2-catalysed reaction.

Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IκBα) by the Inhibitor of kappaB kinase (IKK) that triggers IκBα degradation. Although inhibitor of kappaB beta (IκBβ) is structurally similar to IκBα, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IκBβ with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IκBα.

HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome.

Structural basis for eIF2B inhibition in integrated stress response.

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B.

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity.