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

• DOI: 10.1007/978-1-4939-7634-8_11
• Publication Date: 2018-02-26T08:46:54Z
• Authors: Erich Michel; Olivier Duss; Frédéric H.-T. Al...
• Keywords: Translational regulation; Chloramphenicol acetyltransferase; Eukaryotic translation

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.