title: Ribosome RNA assembly intermediates visualized in living cells.
abstract: In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes.
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: McGinnis
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Weeks
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S