title: Modular Assembly of the Bacterial Large Ribosomal Subunit.
abstract: The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.
authors.affiliation: Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Davis
authors.affiliation: National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Yong Zi
authors.initials: YZ
authors.lastname: Tan
authors.affiliation: National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.affiliation: National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Clinton S
authors.initials: CS
authors.lastname: Potter
authors.affiliation: Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address: dlyumkis@salk.edu.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Lyumkis
authors.affiliation: Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address: jrwill@scripps.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Williamson
mesh_terms: D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D055503:Protein Multimerization; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D012329:RNA, Bacterial; D012335:RNA, Ribosomal
keywords: 50S subunit; RNA folding; Ribosome assembly; macromolecular assembly; quantitative mass spectrometry; single-particle cryo-electron microscopy