A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli
Dipali G Sashital,
Candacia A Greeman,
Dmitry Lyumkis,
Clinton S Potter,
Bridget Carragher,
James R Williamson
Affiliations
Dipali G Sashital
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States
Candacia A Greeman
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States
Dmitry Lyumkis
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States; National Resource for Automated Molecular Microscopy, Scripps Research Institute, La Jolla, United States
Clinton S Potter
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States; National Resource for Automated Molecular Microscopy, Scripps Research Institute, La Jolla, United States
Bridget Carragher
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States; National Resource for Automated Molecular Microscopy, Scripps Research Institute, La Jolla, United States
James R Williamson
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States; Department of Chemistry, Scripps Research Institute, La Jolla, United States; Skaggs Institute for Chemical Biology, Scripps Research Institute, La Jolla, United States
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.
