CATALYTIC AND STRUCTURAL ROLES OF NUCLEOBASES AND METAL IONS IN SMALL RIBOZYMES: NEW INSIGHTS FROM CALCULATIONS AND COMPARISON TO EXPERIMENTS
Open Access
- Author:
- Veeraraghavan, Narayanan
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 27, 2011
- Committee Members:
- Prof Sharon Hammes Schiffer, Dissertation Advisor/Co-Advisor
Philip C. Bevilacqua, Committee Chair/Co-Chair
Sharon Hammes Schiffer, Committee Chair/Co-Chair
William George Noid, Committee Member
Andrey S Krasilnikov, Committee Member
Reka Z Albert, Committee Member - Keywords:
- NLPB
reverse GU wobble
wobble
ribozyme
RNA
molecular dynamics
HDV - Abstract:
- The hepatitis delta virus (HDV) ribozyme is a small self-cleaving RNA with a compact tertiary structure and buried active site that functions to cleave an internal phosphodiester bond and linearize concatemers during the rolling-circle genome replication of the virus. Crystal structures of the ribozyme have been solved in both precleaved and product forms and reveal an intricate network of interactions that conspire to catalyze bond cleavage. In addition, extensive biochemical studies have been performed to work out a mechanism for bond cleavage in which C75 and a magnesium ion catalyze the reaction by general acid-base chemistry. Some questions that have remained unclear in this and other ribozymes is the nature of long-distance communication between peripheral regions of the RNA and the buried active site, the roles of metal ions within and near the active site, and the structural effects of varying pH and its consequent impact on catalysis. We performed molecular dynamics simulations and non-linear Poisson Boltzmann electrostatic calculations on the HDV ribozyme in the precleaved and product forms and assessed the above probing questions. Overall, these studies indicate that small functional RNAs have the potential to communicate interactions over long distances, and that wild-type RNAs may have evolved ways to prevent such interactions from interfering with catalysis. We identified a rare reverse G•U wobble close to the active site and show that this motif in conjunction with surrounding phosphates forms an anionic pocket in the active site. This anionic pocket likely serves to bind metal ions and to help shift the pKa of the catalytic nucleobase, C75. Thus, the reverse G•U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme. We further identified two types of Mg2+ ions associated with the ribozyme, chelated and diffuse, at the reverse and standard G•U wobbles, respectively, which appear to contribute to catalysis and stability, respectively. These two metal ion sites exhibit relatively independent behavior. Protonation of C75 was observed to locally organize the active site in a manner that facilitates the catalytic mechanism, in which C75+ acts as a general acid and Mg2+ as a Lewis acid. The simulations also indicated that the overall structure and thermal motions of the ribozyme are not significantly influenced by the catalytic Mg2+ interaction or C75 protonation. This is suggestive of a reaction pathway of the ribozyme dominated by small local motions at the active site rather than large-scale global conformational changes. These results are consistent with a wealth of experimental data and pave the way for a greater understanding of structure-function relationships and the role of metal ions in RNA.