Dynamic architecture of a minimal RNA polymerase II open promoter complex.

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.

Personalized Progressive Addition Lenses: Correlation between Performance and Design.

PURPOSE: A continuous set of personalized designs (design space) for progressive addition lenses (PALs) is investigated. The main goals are (1) to study how the subjects' perception of a personalized design depends on its position in the design space and (2) to compare the performance of personalized PALs to a conventional PAL with a fixed design. METHODS: In a double-blind study, 51 subjects compared Rodenstock Impression FreeSign 3, which is a family of PALs with a continuously controllable personalized design, and Rodenstock Progressiv Life Free, which is a conventional PAL with a single fixed design. The positions and sizes of viewing zones and the softness of gradients of mean power and astigmatism of personalized lenses were customized to individual viewing preferences. These designs were represented as points in a design space comprising a continuum of PAL designs. Subjective ratings and experimental measurements were used to study viewing zone widths, blur gradient smoothness, amount of distortion, the feeling of safety during motion, and overall wearing comfort. RESULTS: (1) Far viewing zone width (experiments and ratings), near viewing zone width (experiments), blur gradient smoothness, and the amount of distortion (ratings) were significantly dependent on the position of the personalized lens design in the design space. This was consistent with the structure of the design space. (2) 82% of the subjects chose personalized lenses as their favorite. Most subjects reported higher wearing comfort and tolerability with personalized lenses than with conventional lenses. CONCLUSIONS: The designs of the tested personalized lenses were perceived by the subjects as intended. This is a prerequisite to the successful customization of PALs to individual wearing preferences. Possible reasons for the preference of the tested personalized lenses are the optimization with respect to individual wearing conditions and the personalization.

Bayesian-inference-based fluorescence correlation spectroscopy and single-molecule burst analysis reveal the influence of dye selection on DNA hairpin dynamics.

Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures.

A nano-positioning system for macromolecular structural analysis.

Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.

Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex.

Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop beta10-beta11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation-elongation transition.

DNA based molecular motors.

Most of the essential cellular processes such as polymerisation reactions, gene expression and regulation are governed by mechanical processes. Controlled mechanical investigations of these processes are therefore required in order to take our understanding of molecular biology to the next level. Single-molecule manipulation and force spectroscopy have over the last 15 years been developed into extremely powerful techniques. Applying these techniques to the investigation of proteins and DNA molecules has led to a mechanistic understanding of protein function on the level of single molecules. As examples for DNA based molecular machines we will describe single-molecule experiments on RNA polymerases as well as on the packaging of DNA into a viral capsid-a process that is driven by one of the most powerful molecular motors.

Application of the nano-positioning system to the analysis of fluorescence resonance energy transfer networks.

Single-molecule fluorescence resonance energy transfer (sm-FRET) has been recently applied to distance and position estimation in macromolecular complexes. Here, we generalize the previously published Nano-Positioning System (NPS), a probabilistic method to analyze data obtained in such experiments, which accounts for effects of restricted rotational freedom of fluorescent dyes, as well as for limited knowledge of the exact dye positions due to attachment via flexible linkers. In particular we show that global data analysis of complete FRET networks is beneficial and that the measurement of FRET anisotropies in addition to FRET efficiencies can be used to determine accurately both position and orientation of the dyes. This measurement scheme improves localization accuracy substantially, and we can show that the improvement is a consequence of the more precise information about the transition dipole moment orientation of the dyes obtained by FRET anisotropy measurements. We discuss also rigid body docking of different macromolecules by means of NPS, which can be used to study the structure of macromolecular complexes. Finally, we combine our approach with common FRET analysis methods to determine the number of states of a macromolecule.