SummaryTFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation.

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimationbased Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

Förster resonance energy transfer (FRET) can be used to measure distances and infer structures at the molecular level. However, the flexible linkers with which the fluorophores are attached to a macromolecule introduce a lack of knowledge. Both the dye’s geometry and kinetics give rise to uncertainties. Whereas the impact of the geometry is already well understood, the real extent of the kinetics has not been investigated thoroughly. Here, we present a single-molecule (sm)­FRET theory that defines the kinetics of dye movements in a complete form. We introduce a formal nomenclature and provide a recipe for the calculation of the corresponding FRET efficiency. We further analyze experimental data in order to obtain parameters characterizing the geometry and kinetics of the FRET dyes and use them to resimulate the FRET efficiencies by diffusion of fluorophore and linker movement. We show in a real case scenario of dye molecules attached to dsDNA that when making geometrical and kinetic assumptions commonly used in the FRET community one obtains results differing from the experimental data. In contrast, our stochastic simulations taking kinetic parameters from experiments into account reproduce the correct FRET efficiencies. Furthermore, we present a method enabling us to classify the kinetics of the dyes by investigating single realizations of the simulated transfer process. The results support our notion that the common kinetic assumptions are not appropriate over the whole range of distances inferred by FRET even for the simple situation of dyes attached to DNA where few interactions occur.