Mutational Analysis of sRNA-mRNA Base Pairing by Electrophoretic Mobility Shift Assay

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearch

Abstract

Small regulatory RNAs (sRNAs) in bacteria often act by base pairing to mRNAs. Direct interactions between an sRNA and its target mRNA can be investigated by electrophoretic mobility shift assay. In this assay, regions engaged in base pairing are analyzed by introducing mutations in one of the RNAs that prevent sRNA–mRNA complex formation, followed by the introduction of complementary mutations in its partner RNA that restore base pairing. Here, we describe the design of a mutational strategy used to analyze the base pairing between two CU-rich regions of the sRNA Rli22 and the AG-rich Shine-Dalgarno region of the mRNA oppA in Listeria monocytogenes. The protocol can be employed for mutational studies of base pairing between any sRNA and its mRNA target(s).

Original languageEnglish
Title of host publicationBacterial Regulatory RNA : Methods and Protocols
EditorsVéronique Arluison, Claudio Valverde
PublisherHumana Press
Publication date2018
Pages165-176
ISBN (Print)978-1-4939-7633-1
ISBN (Electronic)978-1-4939-7634-8
DOIs
Publication statusPublished - 2018
SeriesMethods in Molecular Biology
Volume1737
ISSN1064-3745

Keywords

  • Base pairing
  • Electrophoretic mobility shift assay
  • Regulatory sRNA
  • Target mRNA
  • Electrophoretic Mobility Shift Assay/methods
  • Listeria monocytogenes/genetics
  • RNA, Messenger/genetics
  • DNA Mutational Analysis/methods
  • Base Pairing
  • Base Sequence
  • Protein Binding
  • Transcription, Genetic
  • RNA, Small Untranslated/genetics
  • Mutation
  • Gene Expression Regulation, Bacterial
  • Nucleic Acid Conformation
  • RNA, Bacterial/genetics

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