title: A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli.
abstract: Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3' domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3'-domain is unanchored and the 5'-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: Dipali G
authors.initials: DG
authors.lastname: Sashital
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: Candacia A
authors.initials: CA
authors.lastname: Greeman
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Lyumkis
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: Clinton S
authors.initials: CS
authors.lastname: Potter
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.affiliation: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States.
authors.forename: James R
authors.initials: JR
authors.lastname: Williamson
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D016624:Negative Staining; D011487:Protein Conformation; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D029968:Escherichia coli Proteins
keywords: 30S subunit; E. coli; RimP; assembly factors; biochemistry; biophysics; electron microscopy; quantitative mass spectrometry; ribosome assembly; structural biology