Super-Resolution Fluorescence Microscopy of Transcription Sites in E. Coli
BIOPHYSICAL JOURNAL 102:3 (2012) 229A-229A
T7 RNA polymerase functions in vitro without clustering.
PLoS One 7:7 (2012) e40207
Abstract:
Many nucleic acid polymerases function in clusters known as factories. We investigate whether the RNA polymerase (RNAP) of phage T7 also clusters when active. Using 'pulldowns' and fluorescence correlation spectroscopy we find that elongation complexes do not interact in vitro with a K(d)<1 µM. Chromosome conformation capture also reveals that genes located 100 kb apart on the E. coli chromosome do not associate more frequently when transcribed by T7 RNAP. We conclude that if clustering does occur in vivo, it must be driven by weak interactions, or mediated by a phage-encoded protein.DAOSTORM: an algorithm for high- density super-resolution microscopy.
Nat Methods 8:4 (2011) 279-280
Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis.
Biophys J 100:6 (2011) 1568-1577
Abstract:
Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneity arises from dynamic processes or from the coexistence of several static structures. To this end, we introduce burst variance analysis (BVA), a method that detects dynamics by comparing the standard deviation of FRET from individual molecules over time to that expected from theory. Both simulations and experiments on DNA hairpins show that BVA can distinguish between static and dynamic sources of heterogeneity in single-molecule FRET histograms and can test models of dynamics against the observed standard deviation information. Using BVA, we analyzed the fingers-closing transition in the Klenow fragment of Escherichia coli DNA polymerase I and identified substantial dynamics in polymerase complexes formed prior to nucleotide incorporation; these dynamics may be important for the fidelity of DNA synthesis. We expect BVA to be broadly applicable to single-molecule FRET studies of molecular structure and to complement approaches such as probability distribution analysis and fluorescence correlation spectroscopy in studying molecular dynamics.Improved temporal resolution and linked hidden Markov modeling for switchable single-molecule FRET.
Chemphyschem 12:3 (2011) 571-579