title: Fluorescence-based monitoring of ribosome assembly landscapes.
abstract: BACKGROUND
  Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control.
  
  
  RESULTS
  We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution.
  
  
  CONCLUSIONS
  This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.
authors.affiliation: Molecular Microbiology, University of Konstanz, Constance, 78457, Germany. rainer.nikolay@Charite.de.|Current address: Institute of Medical Physics and Biophysics, Charité-Universitaetsmedizin Berlin, Berlin, 10117, Germany. rainer.nikolay@Charite.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Nikolay
authors.affiliation: Molecular Microbiology, University of Konstanz, Constance, 78457, Germany. renate.schloemer@uni-konstanz.de.
authors.forename: Renate
authors.initials: R
authors.lastname: Schloemer
authors.affiliation: Screening Center Konstanz, University of Konstanz, Constance, 78457, Germany. silke.mueller@uni-konstanz.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Molecular Microbiology, University of Konstanz, Constance, 78457, Germany. elke.deuerling@uni-konstanz.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D005470:Fluorometry / Q000379:methods*; D055786:Gene Knockout Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D016254:Mutagenesis, Insertional; D055503:Protein Multimerization / Q000187:drug effects; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*; D000097573:Red Fluorescent Protein
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012269:Ribosomal Proteins; D000097573:Red Fluorescent Protein; C028546:ribosomal protein L1; C036004:ribosomal protein S15