Eukaryotic ribosome assembly requires the coordinated processing and extensive remodeling of pre-rRNAs. During late nuclear maturation of the 60S subunit, sequential removal of the internal transcribed spacer 2 (ITS2) is initiated by endonucleolytic cleavage at site C2 by the conserved Las1 nuclease. Las1 acts together with the kinase Grc3 and the Rix1 complex to form the Rixosome, which also functions in transcriptional regulation. However, the assembly of the Rixosome, its recruitment to pre-ribosomes, and its activation for ITS2 cleavage remain unclear. Here, we present cryo-EM structures of the human LAS1 complex, two structures of the isolated Rixosome and nine transition states of Rix1-bound pre-60S particles from Schizosaccharomyces pombe. These structures reveal a dynamic Rixosome architecture in which the heterotetrameric Las1 complex engages one or two copies of the Rix1 complex. Rix1 binding is highly flexible in the human Rixosome but rigid in the yeast complex. The isolated yeast Rixosome remains inactive, but binding to the pre-60S particle triggers a structural rearrangement that allows for substrate engagement and activation of the nuclease. Together, our results define the dynamic architecture of the Rixosome and provide a structural framework for ITS2 processing during nuclear maturation of the eukaryotic 60S ribosomal subunit.
M. Thoms, Jing-Yi Zhu, Xia-Ying Hong et al.· bioRxiv· 0 citations
mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3’-5’ helicase activity and pulling on the mRNA from its 3’ end.
L. Chitoiu, T. Denk, Martin B. D. Müller et al.· bioRxiv· 0 citations
The opportunistic pathogen Pseudomonas aeruginosa ensures its survival by forming mechanically and chemically resistant biofilms, with cationic exopolysaccharide Pel as an abundant constituent of the structural matrix. Despite its biomedical relevance, the mechanisms of Pel synthesis and secretion via the trans-envelope protein machinery are not understood. Here, we examine the structure of the outer membrane export complex PelBC embedded in synthetic nanodiscs and polymer-extracted particles. Both environments preserve the unique architecture of the complex, where the β-barrel PelB is capped with the dodecameric ring of PelC lipoproteins. Cryogenic electron microscopy shows that the polymer-extracted PelB β-barrel is tightly associated with phospholipids and lipid A molecules, and the membrane-facing PelC ring may stabilize lipids of the periplasmic leaflet in defined positions. All-atom molecular dynamics simulation of PelBC in the asymmetric outer membrane of P. aeruginosa corroborate the structural findings and visualize how the essential C-terminal helix of PelC forms multiple electrostatic contacts with the periplasmic leaflet of the outer membrane. Those interactions reduce the lateral mobility of the lipids, stabilize the position of the ring at the interface and may guide folding and assembly of the polysaccharide export machinery. Highlights The PelBC complex is visualized in nanodiscs and polymer-extracted particles The architecture of PelBC is not affected by the chosen membrane mimetics Structure-based molecular dynamics simulations validate PelBC:lipid interactions Lipid mobility in the outer membrane is hindered by the embedded PelBC complex
Cristian Rosales-Hernandez, Marius Benedens, Julien Reißmann et al.· bioRxiv· 0 citations
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