Gene machines

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.

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.

Abstract:

Switchable FRET is the combination of single-molecule Förster resonance energy transfer (smFRET) with photoswitching, the reversible activation and deactivation of fluorophores by light. By photoswitching, multiple donor-acceptor fluorophore pairs can be probed sequentially, thus allowing observation of multiple distances within a single immobilized molecule. Control of the photoinduced switching rates permits adjustment of the temporal resolution of switchable FRET over a wide range of timescales, thereby facilitating application to various dynamical biological systems. We show that fast total internal reflection (TIRF) microscopy can achieve measurements of two FRET pairs with 10 ms temporal resolution within less than 2 s. The concept of switchable FRET is also compatible with confocal microscopy on immobilized molecules, providing better data quality at high temporal resolution. To identify states and extract their transitions from switchable FRET time traces, we also develop linked hidden Markov modeling (HMM) of both FRET and donor-acceptor stoichiometry. Linked HMM successfully identifies transient states in the two-dimensional FRET-stoichiometry space and reconstructs their connectivity network. Improved temporal resolution and novel data analysis make switchable FRET a valuable tool in molecular and structural biology.