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.In recent years, high-resolution structural models of large macromolecular complexes such as the ribosome 1 , the RecBCD helicase 2 or RNA polymerases 3,4 have been obtained using X-ray crystallography. Although these structures provide detailed insight into the molecular architecture of complex biological systems, the position of flexible domains can usually not be determined because of averaging effects.Single-molecule methods, on the other hand, provide the possibility of directly obtaining structural information because they allow the study of real-time conformational changes of macromolecular complexes 5 . In combination with fluorescence resonance energy transfer (FRET) 6 , a technique that has been termed a molecular ruler 7 , one can in principle measure distances within a macromolecule in real-time. However, because of experimental problems such as variations in quantum yield 8 or dependence of FRET on the orientations of the two dye molecules 9 , there are few examples in the literature of quantitative distance measurements 8,10-12 or position determination 13-16 using single-pair FRET (sp-FRET). Instead, these data are more often interpreted in a qualitative fashion monitoring conformational changes and length increases or decreases [17][18][19][20] .Using triangulation of several FRET distance measurements, it is possible to determine a previously unknown position [13][14][15][16][21][22][23] . Although these experiments are able to estimate the most likely position, they do not show how existing experimental uncertainties might affect the position determined. Therefore, these positions must be interpreted with great caution because one has no information about the experimental accuracy. In principle, one can conduct control measurements that provide validity tests of the position determined 14 , but to arrive at a quantitative technique, experimental uncertainties must be taken into account.Here we used bayesian parameter estimation 21 , a probabilitybased analysis method, to compute the three-dimen...

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 β10-β11 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.

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.