Home » Key Scientific Articles » Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria

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

Significance Statement

This works demonstrates the implementation of fluorescence complementation to observe in vivo RNA-protein interactions in prokaryotes between a small RNA (sRNA) and a protein.  This work describes the construction of a functional regulatory sRNA tagged with the RNA binding domain (BD) of the MS2 viral coat protein. This MS2 BD tag acts as an intermediate to effectively recruit the C-terminal domain of the yellow fluorescent protein (YFP) to the sRNA. A fluorescent signal is detected when the interacting protein is tagged with the N terminal domain of YFP.  This work demonstrates that the fluorescence of the signal correlates to the affinity of the RNA-protein interaction. Specifically, we have demonstrated that defective mutant variants of the RNA binding protein display lower levels of fluorescence per cell. This work could be adapted to other sRNA-protein interactions and could serve as an effective tool for the study of these regulatory interactions in vivo.

Figure Legend

Representation of the three fusion molecules interacting to create a functional Yellow Fluorescent Protein: The hairpins represent the RNA fusion with purple region representing the sRNA and the brown region representing the binding domain for the viral coat protein. The protein of interest (magenta ovals) and the viral coat protein (brown square) are fused to the N and C terminal fragments of the Yellow Fluorescent Protein (respectively). When the two proteins bind to their respective binding domain on the RNA fusion, the fragments refold into a functional protein and produce a fluorescent signal.

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

 

 

 

 

 

 

 

 

 

 

 

Journal Reference

Gelderman G1, Sivakumar A, Lipp S, Contreras L. Biotechnol Bioeng. 2015 Feb;112(2):365-75.

The University of Texas at Austin, McKetta Department of Chemical Engineering 200 E. Dean Keeton St. Stop C0400 Austin, Texas, USA 78712.

 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.

© 2014 Wiley Periodicals, Inc.

Go To Biotechnology and Bioengineering