title: In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates.
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 in vivo. Quantitative (14)N/(15)N 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.
authors.affiliation: Cell, Molecular, and Developmental Biology and Biophysics Program, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218-2685, USA.
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Clatterbuck Soper
authors.forename: Romel P
authors.initials: RP
authors.lastname: Dator
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: D000107:Acetylation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D055029:Inverted Repeat Sequences; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D059367:RNA Cleavage / Q000528:radiation effects; D059370:RNA Folding; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D012336:RNA, Ribosomal, 16S; C092748:RbfA protein, E coli; D012269:Ribosomal Proteins; C436973:RimM protein, E coli