Poster abstracts

Poster number 10 submitted by Mark Ditzler

Investigating the Physical Origin of Molecular Heterogeneity in the Hairpin Ribozyme

Mark A. Ditzler (Biophysics University of Michigan), David Rueda (Chemistry Wayne State University), Jingjie Mo, Kristina Hakansson, Nils G. Walter (Chemistry University of Michigan)

Abstract:
Single-molecule techniques provide unique insights into the nature of biologically important macromolecules by revealing both s hort-lived intermediates and weakly populated sub-populations not observed in ensemble measurements. Consequently, molecular heterogeneity has been detected for a variety of systems at the single molecule level. For the hairpin ribozyme, analysis of single-molecule fluorescen ce resonance energy transfer (FRET) time trajectories reveals the presence of persistent heterogeneity in global (un)docking dynamics (1, 2). At least four folding sub-populations have been detected in the minimal hairpin ribozyme. The folding sub-populations are distinguishe d from each other based on their undocking kinetics. The different undocking behaviors persist for individual molecules over long periods of time and through multiple cycles of docking and undocking, giving rise to a so-called "memory effect"(1).
In this work, we characterize the physical origin of the heterogeneous dynamics of the hairpin ribozyme by correlating the heterogeneity observed at the single-molecule level with ensemble measurements. We used native polyacrylamide gel electrophoresis (PAGE) to separate two previously observed folding populations of the hairpin ribozyme (3). Single-molecule and time-resolved ensemble FRET reveal that the gel mobility of the two populations is primarily determined by the molecular heterogeneity observed at the single-molecule level. Chemical and enzymatic foot-printing reveals a shared secondary structure between the two populations. Additionally, native PAGE assays demonstrate that denaturation/refolding alone is not sufficient to inter-convert the two populations, indicating that the ribozyme may be covalently modified. We have applied fourier transform ion cyclotron resonance mass spectrometry to search for covalent modifications of the ribozyme. Our mass spectrometry results do not support the presence of covalent modifications that change the mass. Therefore, our current results are most consistent with models in which the "memory effect" either arises from a mass-neutral covalent modification or from an extraordinarily stable alternate tertiary fold.

References:
1. Zhuang, X., et al., Science, 2002. 296(5572): p. 1473-6.
2. Rueda, D., et al., Proc Natl Acad Sci U S A, 2004. 101(27): p. 10066-71.
3. Pinard, R., et al., Embo J, 2001. 20(22): p. 6434-42.

Keywords: FRET, Single-molecule, Kinetic Trap