RRM
2007Rustbelt RNA Meeting
RRM
|
|
|
|
|
Talk abstracts
Abstract:
The hairpin ribozyme is an RNA motif that facilitates cleavage and ligation of its own backbone. The catalytic activity and small size of the hairpin ribozyme makes it an attractive model system for understanding structure, dynamics, and function in RNA. Despite numerous structural and biochemical investigations the catalytic mechanism of the ribozyme’s self-cleavage remains unclear. However, multiple experiments do support the proposal that the protonation state of A38 plays a direct role in catalysis. In an attempt to gain insight into the impact of both A38 protonation and global stability on the catalytic activity of the hairpin ribozyme, we employ molecular dynamics (MD) simulations and native gel electrophoresis. We first examine the impact of an in vitro selected “gain of function” mutation at position 39 through both MD simulations and native gel electrophoresis. Our simulations predict improved global stability in this mutant which is validated by our observation that the mutant ribozyme migrates more rapidly during native gel electrophoresis. In order to investigate the impact of A38 protonation on active site architecture, we compare simulations in which A38 is either protonated or deprotonated at the N1 position. Our unprotonated simulations place N1 in close proximity to the nucleophilic 2’-O indicating that A38 may act as a general base in the cleavage reaction, a role that has generally been discounted based on proximities observed in crystal structures of the ribozyme in complex with a non-cleavable substrate analog. Simulations in which A38 is protonated place N1 in close proximity to the 5’-O leaving group which supports the proposal that A38 serves as a general acid in the cleavage reaction. Based on our simulations and previous biochemical and structural data we propose a new mechanism in which A38 acts as both general acid and general base.
Keywords: molecular dynamics, ribozyme, hairpin ribozyme
