Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli

C. Aagaard, G. Rosendahl, M. Dam, T. Powers, S. Douthwaite

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcriptions 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 α-sarcin loop of 23S rRNA.

Original languageEnglish (US)
Pages (from-to)1439-1444
Number of pages6
JournalBiochimie
Volume73
Issue number12
DOIs
StatePublished - 1991
Externally publishedYes

Keywords

  • antibiotic binding
  • rRNA mutagenesis
  • rRNA structure

ASJC Scopus subject areas

  • Biochemistry

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    Aagaard, C., Rosendahl, G., Dam, M., Powers, T., & Douthwaite, S. (1991). Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli. Biochimie, 73(12), 1439-1444. https://doi.org/10.1016/0300-9084(91)90176-2