Poster abstracts

Poster number 64 submitted by Bishnu Paudel

Characterize thermodynamics and kinetics aspects of the structural heterogeneity in the c-di-GMP riboswitch through laser assisted single molecule refolding

Bishnu P. Paudel (Department of Chemistry, Wayne State University, Detroit, MI, 48202), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI, 48202)

Abstract:
RNAs play important biological roles in gene regulation and expression, RNA processing, viral replication and protein synthesis [1-3]. RNA molecules require proper secondary and tertiary structure folding to perform their biological activity [3-4]. The rugged RNA folding potential landscapes may entrap RNAs molecules in deep energy wells leading to the memory effect [5-6]. The c-di-GMP riboswitch has been shown to have population heterogeneity and the memory effect in which the interconversion between subpopulations is not observed. We have used laser assisted single molecule refolding (LASR) to introduce the interconversion between subpopulations of the c-di-GMP riboswitch and to measure the barrier heights of interconversion [6]. Our single molecule data reveals that in absence of the ligand (c-di-GMP), the riboswitch is mostly present in static undocked (sub-population1) and dynamic undocked (sub-population2) in which riboswitch is primarily in an undocked conformation with transient fluctuations to a docked structure. Molecules interconvert between sub-population1 and sub-population2 as the temperature is increased, however the percentage of interconversion even at high temperature (74˚C) is low (≤22%). These two states are separated by large energy barriers which slow interconversion. The entropic contribution is more significant than enthalpy to the interconversion barriers. When the two conformations are separated by native gel, these subpopulations redistribute to equilibrium at higher temperature. The structural elucidation of the memory effect will contribute significantly to understanding the RNA folding landscape.

References:
1.Guo, S., et al., Specific delivery of therapeutic RNAs to cancer cells via the dimerization mechanism of phi29 motor pRNA. Hum Gene Ther, 2005. 16(9): P. 1097-109.
2.Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411(6836):p. 494-8.
3.Pyle, A.M., Metal ions in the structure and function of RNA. J Biol Inorg Chem, 2002. 7(7-8): p. 679-90.
4.Woodson, S.A., Metal ions and RNA folding: a highly charged topic with a dynamic future. Curr Opin Chem Biol, 2005. 9(2): p. 104-9.
5.Zhuang, X. et al., Correlating structural dynamics and function in single ribozyme molecules. Science, 2002. 296(5572): p. 1473-6.
6.Zhao, R., et al., Laser-assisted single-molecule refolding (LASR). Biophys J, 2010.99(6): p. 1925-31.

Keywords: c-di-GMP riboswitch, memory effect, smFRET