Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 degrees C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

Automated summary

Ribosome biogenesis is a complex process that relies on various assembly factors. We investigated this process by studying the impact of heat stress on the late stages of ribosome assembly, which led to the accumulation of 30S precursors. We used mass spectrometry-based proteomics and cryo-electron microscopy to determine the protein contents and structures of these precursors.