Poster abstracts

Poster number 37 submitted by Huizhong Xu

Sequence-dependent folding pathways of single P5ab RNAs

Huizhong Xu (Physics, Carnegie Mellon University), Muwen Kong (MBSB, University of Pittsburgh), Ryan Slack (MBSB, University of Pittsburgh), Krishna Agrawal (Physics, Max Planck Research School), Maumita Mandal (Chemistry, Carnegie Mellon University)

Abstract:
P5ab is a RNA hairpin, i.e. an A-form double helix, which is the basic unit of RNA structure. It is part of the catalytic core in the Tetrahymena thermophila ribozyme. Since its identification by Cech and coworkers, this RNA hairpin has been studied by X-ray crystallography, biochemical methods, temperature jump and single-molecule assays by several groups over the decades [1-4]. It has been previously characterized as a two-state folder in single-molecule studies [3, 4]. Here using single-molecule force spectroscopy with resolutions of sub-nanometer and millisecond, we first identify a fast intermediate state I (rate constant ~ 400 /sec) along the folding pathway of P5ab, suggesting a more complex folding route than initially thought. Then to better characterize state I, a mutant m1 is designed by replacing two G-U wobbles with G-C pairs in the hairpin stem, resulting in a more stable state I (rate constant ~30 /sec), which now makes the hairpin (un)fold hierarchically in two discrete steps. Based on the m1 sequence, a mutant m2 aimed at creating a new intermediate I2 is achieved by insertion of an A-A bulge between state I and the tetraloop. It turns out such mutation also changes the folding cooperativity of the hairpin, which now unfolds all the way to the intermediate I2, bypassing state I. Next we demonstrate in mutant m3 how to stabilize another intermediate I1 by both base insertions and replacements. Finally we reconstructe the free energy landscape profiles from the extension probability under constant force followed by the deconvolution procedure. We present, to our knowledge for the first time, the full sequence-dependent folding landscapes of the RNAs, which demonstrate how single-base mutations in the sequence can drastically alter the folding kinetics, pathways and cooperativity. Such detailed information will be crucial for understanding mechanisms by which molecular motors make genetic decisions based on RNA folding kinetics.

References:
1. J. H. Cate, A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot, T. R. Cech, J. A. Doudna, Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678 (1996).
2. S. K. Silverman, T. R. Cech, Energetics and cooperativity of tertiary hydrogen bonds in RNA structure. Biochemistry 38, 8691-8702 (1999).
3. J. Liphardt, B. Onoa, S. B. Smith, I. Tinoco Jr., C. Bustamante, Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733 (2001).
4. J. Wen, M. Manosas, P. T. X. Li, S. B. Smith, C. Bustamante, F. Ritort, I. Tinoco, Jr., Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys. J. 92, 2996 (2007).

Keywords: P5ab, optical tweezers, free energy landscape