Weis, F. | Search bioRxiv

bioRxiv2018

Structural rearrangement of TFIIS- and TFIIF/TFIIE-like subunits in RNA polymerase I transcription complexes

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-EM structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-electron microscopy structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 [A] resolution that provide additional insight into the functional interplay between the TFIIE/TFIIF-like A49-A34.5 heterodimer and the TFIIS-like subunit A12.2 present in Pol I. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our work suggests a regulatory mechanism of Pol I transcription where the association of the A49-A34.5 heterodimer to Pol I is regulated by subunit A12.2, thereby explaining in vitro biochemical and kinetic data.

molecular biology↗

bioRxiv2017

Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, eIF2B

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the {beta} and {delta} regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

molecular biology↗