A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

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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.

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Dipali G Sashital

Iowa State University, Ames, United States

Competing interests
The authors declare that no competing interests exist.
Candacia A Greeman

Scripps Research Institute, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
Dmitry Lyumkis

Salk Institute for Biological Studies, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
Clinton Potter

Scripps Research Institute, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
Bridget Carragher

Scripps Research Institute, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
James R Williamson

Scripps Research Institute, La Jolla, United States

For correspondence
jrwill@scripps.edu

Competing interests
The authors declare that no competing interests exist.

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