InVivo X-Ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates

SarahF ClatterbuckSoper, RomelP Dator, PatrickA Limbach, SarahA Woodson

Research output: Contribution to journalArticle

Abstract

Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E.coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription invivo. Quantitative 14N/15N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly. •Pre-30S ribosomes were visualized insitu by X-ray footprinting on whole cells•Pre-30S structures reveal widespread refolding of pre-rRNA late in assembly•RimM and RbfA change folding paths of the 16S 5' and 3' domains•Quantitative mass spectrometry correlates protein acetylation with rRNA structure.

Original languageEnglish (US)
Pages (from-to)506-516
Number of pages11
JournalMolecular Cell
Volume52
Issue number4
DOIs
StatePublished - Nov 21 2013

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Ribosomes
X-Rays
Acetylation
Proteins
Mass Spectrometry
RNA Folding
RNA
Ribosome Subunits
Synchrotrons
RNA Precursors
Hydroxyl Radical
Cytoplasm
Escherichia coli
Bacteria

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Medicine(all)

Cite this

InVivo X-Ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates. / ClatterbuckSoper, SarahF; Dator, RomelP; Limbach, PatrickA; Woodson, SarahA.

In: Molecular Cell, Vol. 52, No. 4, 21.11.2013, p. 506-516.

Research output: Contribution to journalArticle

ClatterbuckSoper, SarahF ; Dator, RomelP ; Limbach, PatrickA ; Woodson, SarahA. / InVivo X-Ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates. In: Molecular Cell. 2013 ; Vol. 52, No. 4. pp. 506-516.
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