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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.

In recent years, high-resolution structural models of large macromolecular complexes such as the ribosome, the RecBCD helicase or RNA polymerases 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.




© Nature Methods 5, 965 - 971 (2008)




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. In combination with fluorescence resonance energy transfer (FRET), a technique that has been termed a molecular ruler, one can in principle measure distances within a macromolecule in real-time. However, because of experimental problems such as variations in quantum yield or dependence of FRET on the orientations of the two dye molecules, there are few examples in the literature of quantitative distance measurements or position determination 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.

Using triangulation of several FRET distance measurements, it is possible to determine a previously unknown position. 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, but to arrive at a quantitative technique, experimental uncertainties must be taken into account.

Here we used bayesian parameter estimation, a probability-based analysis method, to compute the three-dimensional probability density function for the position of a so-called 'antenna dye molecule' (ADM) attached to a flexible domain within a macromolecular complex. By determining its position, we inferred the position of the flexible domain. We measured the FRET efficiencies between the ADM and several 'satellite dye molecules' (SDMs) attached to known positions. Within the bayesian framework we were able to account for errors (due to orientational effects) in the Förster radii determined, errors in the measured FRET efficiencies and uncertainties in the SDM positions (due to the attachment of the SDMs by means of flexible linkers). Moreover, we were able to account for geometric constraints such as occluded volumes and maximum distances. From the calculated probability density function, we directly obtained the most likely ADM position and the associated three-dimensional credibility regions reflecting the experimental uncertainties. This method has similarities with the Global Positioning System (GPS), and we therefore termed this technique 'Nano Positioning System' (NPS).

As an example, we used NPS to study the position of nascent RNA in yeast RNA polymerase II (Pol II) elongation complexes. We had previously used sp-FRET–based triangulation to follow the path of the nascent RNA and shown that RNA leaves the active-site cleft of the polymerase through the so-called exit tunnel and then follows a path across the dock domain.

Here we applied NPS to determine the position of an ADM attached to the 3' or 5' end of a 29-nucleotide (nt)-long RNA in a Pol II elongation complex. Moreover, we have investigated how the latter position is influenced by the presence of transcription factor IIB (TFIIB). The results allow us to explain discrepancies between recent RNA chemical cross-linking experiments and single-molecule fluorescence data on the position of the RNA, and they have implications for our understanding of the transition from transcription initiation to elongation.

“A nano-positioning system for macromolecular structural analysis”
Adam Muschielok, Joanna Andrecka, Anass Jawhari, Florian Brückner, Patrick Cramer, Jens Michaelis
Nature Methods 5, 965 - 971 (2008)

Adam Muschielok

since 2006
PhD student in the group of Prof. Jens Michaelis, LMU Munich

2001 - 2005
Diploma in Physics at the TU Munich

Selected Publication

J. Michaelis, A. Muschielok, J. Andrecka, W. Kügel, and J. Moffitt:
"DNA based molecular motors"
Physics of Life Reviews (2009)

Ioanna Andrecka

since 2009
Postdoctoral research at the LMU München in the group of Prof. Jens Michaelis

2005 - 2009
Doctoral Thesis, Supervisor: Prof. Dr. Jens Michaelis, LMU Munich

2004 - 2005
PhD Studies of Biochemistry and Biophysics, Jagiellonian University of Cracow, Poland

1999 - 2004
Interdisciplinary, Individual MSc Studies of Mathematical and Natural Sciences, Jagiellonian University of Cracow, Poland

Selected Publication

Andrecka J., Treutlein B., Arcusa M.A., Muschielok A., Lewis R., Cheung A.C., Cramer P., Michaelis J.:
"Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex"
Nucleic Acid Research 37 (17), 5803-9 (2009)