Concurrent remodeling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase
- Heidelberg University, Germany
- Ludwig-Maximilians-Universität München, Germany
Abstract
Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.
Data availability
Atomic models reported in this study have been deposited in the Protein Data Bank (PDB) and can be retrieved using the following accession codes: 8BVN, 8BVU, 8BVV, 8BVY. Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) and can be retrieved using the following accession codes: 16267, 16272, 16273, 16275, 16276, 16277, 16278. Yeast strains and plasmids are available from the corresponding authors upon request.
Article and author information
Author details
Funding
European Research Council (885711-Human-Ribogenesis)
- Roland Beckmann
Deutsche Forschungsgemeinschaft (HU363/15-2)
- Ed Hurt
European Research Council (ADG 741781 GLOWSOME)
- Ed Hurt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Mitterer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
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