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A nano-positioning system for macromolecular structural analysis

Nature Methods volume 5, pages 965–971 (2008)Cite this article

Abstract

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.

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Figure 1: Single-pair FRET time traces and histograms for RNA 29–Rpb7-S16C complexes.
Figure 2: The concept of fuzzy spheres, presented using the two-dimensional analog, fuzzy circles.
Figure 3: Schematic representation of 'antenna' and 'satellite' dye positions.
Figure 4: Experimental test of NPS: the position of an ADM attached to the 3′ end of the RNA in a Pol II elongation complex.
Figure 5: Computed position of the ADM attached to 5′ end of the 29-nt RNA.

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References

  1. Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002).

    Article  CAS  Google Scholar 

  2. Singleton, M.R. et al. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187–193 (2004).

    Article  CAS  Google Scholar 

  3. Cramer, P., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876 (2001).

    Article  CAS  Google Scholar 

  4. Vassylyev, D.G. et al. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007).

    Article  CAS  Google Scholar 

  5. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    Article  CAS  Google Scholar 

  6. Forster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys-Berlin 437, 55–75 (1948).

    Article  Google Scholar 

  7. Bregeon, D. et al. Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli. Mol. Cell 12, 959–970 (2003).

    Article  CAS  Google Scholar 

  8. Kapanidis, A.N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).

    Article  Google Scholar 

  9. Dale, R.E., Eisinger, J. & Blumberg, W.E. The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. Biophys. J. 26, 161–193 (1979).

    Article  CAS  Google Scholar 

  10. Myong, S. et al. Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 317, 513–516 (2007).

    Article  CAS  Google Scholar 

  11. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    Article  CAS  Google Scholar 

  12. Rothwell, P.J. et al. Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc. Natl. Acad. Sci. USA 100, 1655–1660 (2003).

    Article  CAS  Google Scholar 

  13. Rasnik, I. et al. DNA-binding orientation and domain conformation of the E. coli Rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol. 336, 395–408 (2004).

    Article  CAS  Google Scholar 

  14. Andrecka, J. et al. Single-molecule tracking of mRNA exiting from RNA polymerase II. Proc. Natl. Acad. Sci. USA 105, 135–140 (2008).

    Article  CAS  Google Scholar 

  15. Schröder, G.F. & Grubmüller, H. FRETsg: biomolecular structure model building from multiple FRET experiments. Comput. Phys. Commun. 158, 150–157 (2004).

    Article  Google Scholar 

  16. Margittai, M. et al. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc. Natl. Acad. Sci. USA 100, 15516–15521 (2003).

    Article  CAS  Google Scholar 

  17. Zhuang, X. et al. Correlating structural dynamics and function in single ribozyme molecules. Science 296, 1473–1476 (2002).

    Article  CAS  Google Scholar 

  18. McKinney, S.A. et al. Structural dynamics of individual Holliday junctions. Nat. Struct. Biol. 10, 93–97 (2003).

    Article  CAS  Google Scholar 

  19. Diez, M. et al. Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nat. Struct. Mol. Biol. 11, 135–141 (2004).

    Article  CAS  Google Scholar 

  20. Lewis, R. et al. Conformational changes of a Swi2/Snf2 ATPase during its mechano-chemical cycle. Nucleic Acids Res. 36, 1881–1890 (2008).

    Article  CAS  Google Scholar 

  21. Sivia, D.S. Data Analysis: A Bayesian Tutorial Ch. 1–3, 3–77 (Clarendon Press, Oxford, UK, 1996).

    Google Scholar 

  22. Alber, F. et al. Determining the architectures of macromolecular assemblies. Nature 450, 683–694 (2007).

    Article  CAS  Google Scholar 

  23. Medintz, I.L. et al. A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly. Proc. Natl. Acad. Sci. USA 101, 9612–9617 (2004).

    Article  CAS  Google Scholar 

  24. Radman-Livaja, M. et al. Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes. Proc. Natl. Acad. Sci. USA 102, 3913–3920 (2005).

    Article  CAS  Google Scholar 

  25. Sun, X. et al. Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site. Mol. Cell 24, 569–580 (2006).

    Article  CAS  Google Scholar 

  26. Mekler, V. et al. Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599–614 (2002).

    Article  CAS  Google Scholar 

  27. Jung, C. et al. Simultaneous measurement of orientational and spectral dynamics of single molecules in nanostructured host-guest materials. J. Am. Chem. Soc. 129, 5570–5579 (2007).

    Article  CAS  Google Scholar 

  28. Toprak, E. et al. Defocused orientation and position imaging (DOPI) of myosin V. Proc. Natl. Acad. Sci. USA 103, 6495–6499 (2006).

    Article  CAS  Google Scholar 

  29. Ujvari, A. & Luse, D.S. RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit. Nat. Struct. Mol. Biol. 13, 49–54 (2006).

    Article  CAS  Google Scholar 

  30. Bushnell, D.A. et al. Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 angstroms. Science 303, 983–988 (2004).

    Article  CAS  Google Scholar 

  31. Pal, M., Ponticelli, A.S. & Luse, D.S. The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell 19, 101–110 (2005).

    Article  CAS  Google Scholar 

  32. Jasiak, A.J. et al. Genome-associated RNA polymerase II includes the dissociable RPB4/7 subcomplex. J. Biol. Chem. 283, 26423–26427 (2008).

    Article  CAS  Google Scholar 

  33. Runner, V.M., Podolny, V. & Buratowski, S. The Rpb4 subunit of RNA polymerase II contributes to cotranscriptional recruitment of 3′ processing factors. Mol. Cell. Biol. 28, 1883–1891 (2008).

    Article  CAS  Google Scholar 

  34. Kettenberger, H., Armache, K.J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank V. Dose, D.C. Lamb and C. Bräuchle for discussions as well as S. Waszak for help with the programming. The work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 646, the Center for Nanoscience and the Nanosystems Initiative Munich.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry and Center for Integrated Protein Science München, Ludwig-Maximilians-Universität München, Butenandtstrasse 11, 81377, München, Germany

    Adam Muschielok, Joanna Andrecka, Anass Jawhari, Florian Brückner, Patrick Cramer & Jens Michaelis

  2. Gene Center München, Ludwig-Maximilians-Universität München, Feodor Lynen Strasse 25, 81377, München, Germany

    Anass Jawhari, Florian Brückner & Patrick Cramer

Authors
  1. Adam Muschielok
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  6. Jens Michaelis
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Contributions

A.M. performed calculations, designed analysis, wrote the analysis program and wrote the paper; J.A. performed experiments and wrote the paper; F.B. provided reagents; A.J. performed experiments and provided reagents; P.C. and J.M. designed experiments and wrote the paper.

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Correspondence to Jens Michaelis.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1, Supplementary Methods (PDF 3098 kb)

Supplementary Data

XPLOR files for the ADM position probability density for the position of RNA 1 (with and without structural constraints) and RNA 29 (with and without TFIIB) (ZIP 2250 kb)

Supplementary Software

Compiled Matlab software for NPS including detailed instructions (ZIP 758 kb)

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Muschielok, A., Andrecka, J., Jawhari, A. et al. A nano-positioning system for macromolecular structural analysis. Nat Methods 5, 965–971 (2008). https://doi.org/10.1038/nmeth.1259

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