Abstract
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.