Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria

• Published in: Biotechnology and bioengineering Vol. 112; no. 2; pp. 365 - 375
• Main Authors: Gelderman, Grant, Sivakumar, Anusha, Lipp, Sarah, Contreras, Lydia
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.02.2015; Wiley Subscription Services, Inc
• Subjects: Bacteria; Binding; Biomedical materials; Carbon capture and storage; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescence; fluorescence techniques; Genetic Complementation Test - methods; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Metabolism; Mutation; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Ribonucleic acids; RNA, Bacterial - genetics; RNA, Long Noncoding - chemistry; RNA, Long Noncoding - genetics; RNA, Long Noncoding - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; RNA-protein interactions; Surgical implants; RNA-protein interactions; fluorescence techniques
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.25351

ABSTRACT sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri‐molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA–protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB–CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA–protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. Biotechnol. Bioeng. 2015;112: 365–375. © 2014 Wiley Periodicals, Inc. This work demonstrates the application of a fluorescence technique to study a regulatory RNA‐protein interaction. This figure shows the RNA and protein fusions necessary to produce a fluorescent signal in response to the interaction of the Carbon Storage Regulator in Escherichia coli. In addition, the authors demonstrate how this technique can be used to differentiate differences in binding affinity between RNA‐protein interactions based on the intensity of the fluorescent signal.