title: Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting.
abstract: The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Hulscher
authors.affiliation: Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
authors.forename: Jen
authors.initials: J
authors.lastname: Bohon
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Mollie C
authors.initials: MC
authors.lastname: Rappé
authors.affiliation: Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Sayan
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
authors.forename: Rhijuta
authors.initials: R
authors.lastname: D'Mello
authors.affiliation: Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Sullivan
authors.affiliation: Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Corie Y
authors.initials: CY
authors.lastname: Ralston
authors.affiliation: Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA. Electronic address: swoodson@jhu.edu.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: D018929:Cell Culture Techniques; D004926:Escherichia coli; D017665:Hydroxyl Radical / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000648:ultrastructure*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000648:ultrastructure*; D012270:Ribosomes / Q000737:chemistry / Q000648:ultrastructure*; D013194:Staining and Labeling; D013463:Sulfuric Acid Esters / Q000737:chemistry*
chemical_list: D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D013463:Sulfuric Acid Esters; D017665:Hydroxyl Radical; C007482:dimethyl sulfate
keywords: 4-Thiouridine; Dimethylsulfate; Hydroxyl radical footprinting; RNA structure; Ribosome assembly; Synchrotron X-ray beamline