1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

  1. Dipali G Sashital
  2. Candacia A Greeman
  3. Dmitry Lyumkis
  4. Clinton Potter
  5. Bridget Carragher
  6. James R Williamson  Is a corresponding author
  1. Iowa State University, United States
  2. Scripps Research Institute, United States
  3. Salk Institute for Biological Studies, United States
Research Article
  • Cited 20
  • Views 3,049
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Cite this article as: eLife 2014;3:e04491 doi: 10.7554/eLife.04491

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.

Article and author information

Author details

  1. Dipali G Sashital

    Iowa State University, Ames, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Candacia A Greeman

    Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Dmitry Lyumkis

    Salk Institute for Biological Studies, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Clinton Potter

    Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Bridget Carragher

    Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. 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.

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Publication history

  1. Received: August 24, 2014
  2. Accepted: October 13, 2014
  3. Accepted Manuscript published: October 14, 2014 (version 1)
  4. Version of Record published: November 10, 2014 (version 2)

Copyright

© 2014, Sashital 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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