Rapid in vivo exploration of a 5S rRNA neutral network

Zhengdong D. Zhang, Madhavi Nayar, David Ammons, Joanne Rampersad, George E. Fox

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.

Original languageEnglish (US)
Pages (from-to)181-187
Number of pages7
JournalJournal of Microbiological Methods
Volume76
Issue number2
DOIs
Publication statusPublished - Feb 1 2009
Externally publishedYes

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Keywords

  • 5S rRNA
  • E. coli knockout strains
  • In vivo mutagenesis
  • Neutral network

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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