Investigating the Role of an Extended Hydrogen Bonding Network within the Hairpin Ribozyme Active Site

Abstract

From previous studies involving molecular dynamics (MD) simulations and X-ray crystal structures, a chain of five to seven long-residing, ordered water molecules was discovered within the otherwise solvent-protected active site of the hairpin ribozyme (1,2). This water chain is part of an extended hydrogen bonding network within the active site. This network was proposed to be involved both in proton transfer reactions and in mediating long-range communication between the RNA residues in the active site, features important for ribozyme catalytic activity. Until now, however, the functional relevance of this hydrogen bonding network has not been explored. In order to gain insight into the relationship between the hydrogen bonding network and catalytic activity, we are first using MD simulations to computationally scan for ribozyme variants with disrupted hydrogen bonding networks. Specifically, we are studying the behaviour of the hydrogen bonding network in simulations incorporating single-atom modifications in core residues A38, A10, A9 and U42. The use of such subtle alterations did not result in a consistently disrupted hydrogen bonding network between simulation replicates that are 20 nanoseconds long. Therefore, longer such simulations in addition to simulations involving multiple modifications in parallel and those involving single bulky mutations are now being analyzed. Once suitable sets of modifications are predicted by MD simulation, the modified hairpin ribozymes will be chemically synthesized and kinetically characterized using single molecule fluorescence resonance energy transfer together with functional analyses to dissect which kinetic steps, if any, are affected by disruption of the hydrogen bonding network.(1) Rhodes, M. M.; Reblova, K.; Sponer, J.; Walter, N. G. Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 13380-5.(2) Salter, J.; Krucinska, J.; Alam, S.; Grum-Tokars, V.; Wedekind, J. E. Biochemistry, 2006, 45, 686-700.