Interactions in gene expression networks studied by two-photon fluorescence fluctuation spectroscopy

Area of Science:

• Biophysics
• Molecular Biology
• Microbiology

Background:

• Fluorescence fluctuation techniques are vital for studying biomolecular interactions.
• Protein-nucleic acid complexes are key regulators of gene expression.

Purpose of the Study:

• To review recent applications of two-photon fluorescence fluctuation techniques for studying protein-nucleic acid complexes.
• To address complex stoichiometry and gene regulation mechanisms in vitro and in vivo.

Main Methods:

• Fluorescence (cross) correlation spectroscopy (FCS) for in vitro stoichiometry analysis.
• Two-photon scanning number and brightness (2psN&B) for single-cell fluorescence microscopy in live bacteria.
• Adaptation of 2psN&B for analyzing gene expression from inducible promoters in Bacillus subtilis.

Main Results:

• Stoichiometry of RNA-binding protein L20 and transcriptional repressors CggR and CcpN determined in vitro.
• Quantification of fluorescent protein production from various promoters in hundreds of individual B. subtilis cells.
• In vivo stochastic activity of glycolytic and gluconeogenic gene promoters measured using 2psN&B.

Conclusions:

• Two-photon fluorescence fluctuation techniques effectively characterize protein-nucleic acid interactions and gene regulation.
• 2psN&B analysis provides insights into the stochastic nature of gene expression in live bacteria.
• In vivo and in vitro findings correlate, supporting proposed repression mechanisms.