Poster abstracts
Poster number 96 submitted by Aamir Mir
Revisiting the catalytic mechanism of the hammerhead ribozyme
Aamir Mir (Department of Biochemistry, Purdue University), Ji Chen (Department of Biochemistry, Purdue University), Kyle Robinson (Department of Biochemistry, Purdue University), Emma Lendy, Jaclyn Goodman (Purdue University), David Neau (Department of Chemistry and Chemical Biology, Cornell University, Northeastern Collaborative Access Team, Argonne National Laboratory), Barbara L. Golden (Department of Biochemistry, Purdue University)
Abstract:
The hammerhead ribozyme catalyzes the site specific cleavage of a single phosphodiester bond within its RNA substrate. Originally discovered in plant viroids, thousands of hammerhead-like sequences have been found in all domains of life. The cleavage mechanism of the hammerhead ribozyme has been studied extensively but many questions have persisted. The crystal structure of the ribozyme suggests that G12 is the general base as it is well positioned to activate the 2’-hydroxyl of C17 for an in-line nucleophilic attack on the scissile phosphate. Yet, the pH dependence of the G12A mutant is similar to that of the wild-type ribozyme and it is unclear how G12 is activated for proton transfer. In addition, discrepancies exist between the crystal structure and the biochemical data concerning the role of divalent metals in the catalytic mechanism. The crystal structures do not show a divalent metal within the active site of the hammerhead ribozyme. Biochemical data suggest that divalent metals greatly accelerate the hammerhead reaction rate and appear to interact with the pro-Rp oxygen of the scissile phosphate. We studied different hammerhead mutants using both kinetics and X-ray crystallography to address these discrepancies. Our results suggest that conformational changes and two divalent metals are playing important roles in the catalysis of the hammerhead ribozyme.(1-2) Furthermore, based on the pH-rate profile and the divalent metal specificity switch observed in the G12A mutant, we propose that the imino tautomeric species of A12 serves as the general base in G12A hammerhead ribozyme.
References:
1. Mir, A., Chen, J., Robinson, K., Lendy, E., Goodman, J., Neau, D., and Golden, B. L. (2015) Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction, Biochemistry 54, 6369-6381.
2. Mir, A., and Golden, B. L. (2016) Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue, Biochemistry 55, 633-636.
Keywords: hammerhead, ribozyme, self-cleaving