title: Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.
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.
authors: The University of Texas at Austin, McKetta Department of Chemical Engineering 200 E. Dean Keeton St. Stop C0400 Austin, Texas, USA 78712.
authors: Grant
authors: G
authors: Gelderman
authors: Anusha
authors: A
authors: Sivakumar
authors: Sarah
authors: S
authors: Lipp
authors: Lydia
authors: L
authors: Contreras
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C093587:CsrA protein, E coli; C107123:CsrB RNA, E coli; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D012329:RNA, Bacterial; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins
keywords: RNA-protein interactions; fluorescence techniques