Chapter title |
An Integrated Cell-Free Assay to Study Translation Regulation by Small Bacterial Noncoding RNAs
|
---|---|
Chapter number | 11 |
Book title |
Bacterial Regulatory RNA
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7634-8_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7633-1, 978-1-4939-7634-8
|
Authors |
Erich Michel, Olivier Duss, Frédéric H.-T. Allain, Michel, Erich, Duss, Olivier, Allain, Frédéric H.-T. |
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. |
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