An Integrated Cell-Free Assay to Study Translation Regulation by Small Bacterial Noncoding RNAs

Methods Mol Biol. 2018:1737:177-195. doi: 10.1007/978-1-4939-7634-8_11.

Abstract

Posttranscriptional regulation of gene expression by small noncoding RNAs (sRNAs) is an important control mechanism that modulates bacterial metabolism, motility, and pathogenesis. Using the bacterial carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) system, we here describe an E. coli-based cell-free translation assay that allows a quantitative analysis of translation regulation by ncRNAs and their corresponding translation repressor proteins. The assay quantifies the translation of chloramphenicol acetyltransferase in cell-free expression reactions that contain defined amounts of ncRNA and repressor protein. We demonstrate our protocol with a comparative translation activation analysis of the RsmX, RsmY, and RsmZ sRNAs from Pseudomonas protegens, which reveals a superior efficacy of RsmZ over RsmX and RsmY.

Keywords: Bacterial virulence; Carbon storage regulator; Cell-free expression; Chloramphenicol acetyl transferase; CsrA; Noncoding RNAs; Posttranscriptional regulation; Regulator of secondary metabolism; RsmA; Translation regulation.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Gene Expression Regulation, Bacterial*
  • Protein Biosynthesis*
  • Pseudomonas / genetics*
  • Pseudomonas / growth & development
  • RNA, Bacterial / genetics*
  • RNA, Small Untranslated / genetics*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • RNA, Small Untranslated
  • RNA-Binding Proteins