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 journalArticlepeer-review

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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