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Interactions in gene expression networks studied by two-photon fluorescence fluctuation spectroscopy.
MedLine Citation:
PMID:  23280112     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Fluorescence fluctuation techniques have proven to be extremely useful in the characterization of biomolecular interactions. Here an overview of recent applications of two-photon fluorescence fluctuation to the study of protein-nucleic acid complexes implicated in translational and transcriptional regulation is presented. In particular, the issue of the stoichiometry of the complexes is addressed using fluorescence (cross) correlation spectroscopy (F(C)CS) for the in vitro studies of one RNA-binding protein (the bacterial ribosomal protein L20) and two transcriptional repressors (CggR and CcpN, implicated in the control of the central carbon metabolism in Bacillus subtilis). Then, the application of two-photon scanning number and brightness (2psN&B) analysis of fluorescence microscopy measurements in single cells is presented. Multiple technical aspects related to the adaptation of this method to live bacteria are discussed. This approach was used to count the number of fluorescent protein molecules produced from different inducible promoters in B. subtilis reporter strains, in hundreds of individual cells under both permissive and repressive conditions. We present a case study in which the stochastic activity of glycolytic and gluconeogenic gene promoters could be quantified in vivo by 2psN&B and be related to the repression mechanisms proposed from in vitro studies.
Authors:
Nathalie Declerck; Catherine A Royer
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in enzymology     Volume:  519     ISSN:  1557-7988     ISO Abbreviation:  Meth. Enzymol.     Publication Date:  2013  
Date Detail:
Created Date:  2013-01-02     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0212271     Medline TA:  Methods Enzymol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  203-30     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 Elsevier Inc. All rights reserved.
Affiliation:
Centre de Biochimie Structurale, INSERM U1054, CNRS UMR5048, Université Montpellier 1 and 2, Montpellier, France; Département de Microbiologie, Institut National pour la Recherche Agronomique, Paris, France.
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