The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles. In γ-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF). Here we present single-particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary-phase E. coli cells at 3.0 Å and 7.9 Å resolution, respectively. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site transfer RNA and ribosomal protein bS1. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the messenger RNA as well as A- and P-site tRNAs. RMF facilitates stabilization of a compact conformation of bS1, which together sequester the anti-Shine-Dalgarno sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, the C-terminus of uS2 probes the mRNA entrance channel of the symmetry-related particle, thus suggesting that dimerization inactivates ribosomes by blocking the binding of mRNA within the channel. The back-to-back E. coli 100S arrangement is distinct from 100S particles observed previously in Gram-positive bacteria, and reveals a unique role for bS1 in translation regulation.

Under stress conditions, such as nutrient deprivation, bacteria enter into a hibernation stage, which is characterized by the appearance of 100S ribosomal particles. In , dimerization of 70S ribosomes into 100S requires the action of the ribosome modulation factor (RMF) and the hibernation-promoting factor (HPF). Most other bacteria lack RMF and instead contain a long form HPF (LHPF), which is necessary and sufficient for 100S formation. While some structural information exists as to how RMF and HPF mediate formation of 100S (100S), structural insight into 100S formation by LHPF has so far been lacking. Here we present a cryo-EM structure of the hibernating 100S (100S), revealing that the C-terminal domain (CTD) of the LHPF occupies a site on the 30S platform distinct from RMF Moreover, unlike RMF, the HPF-CTD is directly involved in forming the dimer interface, thereby illustrating the divergent mechanisms by which 100S formation is mediated in the majority of bacteria that contain LHPF, compared to some γ-proteobacteria, such as.

In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).

The ribosome is one of the major targets in the cell for clinically used antibiotics. However, the increase in multidrug resistant bacteria is rapidly reducing the effectiveness of our current arsenal of ribosome-targeting antibiotics, highlighting the need for the discovery of compounds with new scaffolds that bind to novel sites on the ribosome. One possible avenue for the development of new antimicrobial agents is by characterization and optimization of ribosome-targeting peptide antibiotics. Biochemical and structural data on ribosome-targeting peptide antibiotics illustrates the large diversity of scaffolds, binding interactions with the ribosome as well as mechanism of action to inhibit translation. The availability of high-resolution structures of ribosomes in complex with peptide antibiotics opens the way to structure-based design of these compounds as novel antimicrobial agents.