scholarly journals In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer

RNA ◽  
2009 ◽  
Vol 15 (11) ◽  
pp. 2063-2071 ◽  
Author(s):  
M. Huranova ◽  
J. A. Jablonski ◽  
A. Benda ◽  
M. Hof ◽  
D. Stanek ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Sequences ◽  
In Vivo Detection

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals In Vivo Self-Interaction of Nodavirus RNA Replicase Protein A Revealed by Fluorescence Resonance Energy Transfer

2005 ◽  
Vol 79 (14) ◽  
pp. 8909-8919 ◽  
Author(s):  
Billy T. Dye ◽  
David J. Miller ◽  
Paul Ahlquist
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Mitochondrial Membrane ◽  
Rna Viruses ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Replication ◽  
Outer Mitochondrial Membrane

ABSTRACT Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

Sign in / Sign up

Export Citation Format

Share Document