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...

Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5 end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5 end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5-capping enzyme and other RNA processing factors.Pol II ͉ transcription ͉ FRET ͉ triangulation ͉ fluorescence P ol II synthesizes all eukaryotic mRNA and comprises 12 subunits, Rpb1 to Rpb12. An atomic model of Pol II has been obtained by x-ray crystallography (1, 2). Additional studies of Pol II-nucleic acid complexes have given insights into the elongation complex structure and molecular aspects of the transcription mechanism (3-7). Crystallographic analysis detected the position of the nascent RNA within the DNA-RNA hybrid above the active site (positions ϩ1 to Ϫ8, with ϩ1 denoting the nucleotide addition site) and for 2 nt upstream of the hybrid after the point of DNA-RNA strand separation (positions Ϫ9 and Ϫ10) (3, 7). The last-ordered RNA nucleotide is located at the entrance to a tunnel [called the RNA exit channel for the bacterial RNA polymerase (8)], which is formed among the polymerase wall, clamp, and lid. This tunnel leads from the active center cleft to the exterior and was proposed to accommodate exiting RNA (1,3,6,7). Beyond the putative exit tunnel, two prominent surface grooves on either side of the dock domain in principle could further accommodate exiting RNA (1, 9). Groove 1 winds along the base of the clamp toward the Rpb4/7 subcomplex, whereas groove 2 leads along Rpb11 toward Rpb8. Recently, nascent RNA could be crosslinked to Rpb7, providing apparent support for groove 1 (10). RNA beyond position Ϫ10 was present in one of the crystallographic studies of the elongation complex (3) but could not be observed in the tunnel or in the subsequent grooves, suggesting that its interactions, if they exist, are transient and cannot be detected in medium-resolution electron density maps.To study if the nascent RNA indeed exits through the proposed tunnel, and whether it follows a defined surface path beyond the tunnel, we used single-molecule fluorescence experiments. Singleparticle methods prevent the loss of information because of averaging that is inherent to bulk experiments and crystallography (11)(12)(13)(14). Fluorescence resonance energy transfer between two fluorophores [single-pair (sp)-FRET] provides a very sensitive tool for studying distances and conformational changes within biological complexes in the ...

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