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 language | English (US) |
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Pages (from-to) | 1439-1444 |
Number of pages | 6 |
Journal | Biochimie |
Volume | 73 |
Issue number | 12 |
DOIs | |
State | Published - 1991 |
Externally published | Yes |
Keywords
- antibiotic binding
- rRNA mutagenesis
- rRNA structure
ASJC Scopus subject areas
- Biochemistry