The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.alternating-laser excitation | conformational dynamics | fidelity checkpoints | Klenow fragment | fingers-closing

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.

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

We form artificial lipid bilayers suitable for single-molecule fluorescence microscopy by contacting an aqueous droplet with a hydrogel support immersed in a solution of lipid in oil. Our results show that droplet on hydrogel bilayers (DHBs) have high lipid mobilities, similar to those observed in unsupported lipid bilayers. DHBs are also stable over a period of several weeks. We examine membrane protein diffusion in these bilayers and report a decreased lateral mobility of the heptameric beta-barrel pore-forming toxin alpha-hemolysin versus that of its monomeric precursor. These results corroborate previous models of the alpha-hemolysin insertion mechanism where the monomer binds to the lipid bilayer without insertion.