title: Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli.
abstract: The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous ribosome population, which consequently hinders direct probing of mutant rRNAs. Here, we describe how nonconserved helical regions of plasmid-coded rRNA have been altered in a manner that preserves their secondary structures while creating new sites for primer extension of mutant rRNAs. This facilitates specific biochemical probing of mutagenised rRNA regions despite the background of wild-type molecules. Four priming sites have been made to investigate the structural effects of mutations in the GTPase centre, helix 1200-1250, the peptidyl transferase region and the alpha-sarcin loop of 23S rRNA.
authors.affiliation: Department of Molecular Biology, Odense University, Denmark.
authors.forename: C
authors.initials: C
authors.lastname: Aagaard
authors.forename: G
authors.initials: G
authors.lastname: Rosendahl
authors.forename: M
authors.initials: M
authors.lastname: Dam
authors.forename: T
authors.initials: T
authors.lastname: Powers
authors.forename: S
authors.initials: S
authors.lastname: Douthwaite
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001483:Base Sequence; D004352:Drug Resistance, Microbial; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010458:Peptidyl Transferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015347:RNA Probes; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D015347:RNA Probes; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D004917:Erythromycin; D010458:Peptidyl Transferases; D020558:GTP Phosphohydrolases