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Chromatographic Purification of Highly Active Yeast Ribosomes
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Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.

Emmanuel Giudice1, Sylvie Georgeault1, Régis Lavigne2,3

  • 1Univ Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR6290, 35000 Rennes, France.

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|February 25, 2023
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Heat stress reveals novel 30S ribosomal subunit precursors. Researchers identified 21S ribosomal particles, key intermediates in ribosome biogenesis, using advanced proteomic and cryo-EM techniques.

Keywords:
21SDnaKcryo-EMheat shockribosome assembly

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Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Ribosome biogenesis is a complex process involving numerous assembly factors.
  • Previous studies often deleted or depleted assembly factors to study intermediates.
  • Heat stress offers an alternative method to investigate ribosome assembly pathways.

Purpose of the Study:

  • To identify authentic ribosome assembly intermediates.
  • To explore the impact of heat stress on 30S ribosomal subunit biogenesis.
  • To characterize the protein content and structure of transiently accumulated ribosomal particles.

Main Methods:

  • Inducing heat stress (45 °C) to affect late stages of 30S subunit biogenesis.
  • Constructing strains with affinity tags on 30S ribosomal proteins.
  • Purifying 21S ribosomal particles using affinity tags.
  • Employing mass spectrometry-based proteomics for protein quantification.
  • Utilizing cryo-electron microscopy (cryo-EM) for structural analysis.

Main Results:

  • Heat stress at 45 °C transiently accumulates 21S ribosomal particles, identified as 30S precursors.
  • Reduced levels of DnaK chaperone proteins are linked to this accumulation.
  • Proteomics and cryo-EM determined the protein composition and structure of these 21S particles.

Conclusions:

  • Heat stress is a valuable tool for uncovering ribosome assembly intermediates.
  • 21S ribosomal particles represent authentic precursors in 30S subunit biogenesis.
  • This study provides insights into the dynamic process of ribosome assembly under stress conditions.