Poster abstracts

Poster number 57 submitted by Ruochuan Huang

Effects of cellular concentrations of nucleotide diphosphate-chelated Mg2+ on RNA and DNA enzymes

Ruochuan Huang (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Ryota Yamagami (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802)

Abstract:
Our recent studies demonstrate that cellular concentrations of weakly amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation.1 However, the effects of other metabolites that have an affinity for Mg2+ are not yet clarified. This study focuses on the effect of nucleotide-chelated Mg2+ since nucleotides also comprise a great part of metabolome in vivo (37 mM total).2 Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.5 mM Mgfree2+. We tested the self-cleaving activity in hammerhead ribozyme (HH16) in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium (NDPCM) promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc. Additionally, we found similar stimulatory effects for a DNAzyme. Thus, rate stimulatory effects appear to be general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

References:
1. Yamagami R, Bingaman JL, Frankel EA, Bevilacqua PC. 2018. Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis. Nat Commun 9: 2149
2. Bennett, B. D., Kimball, E. H., Gao, M., Osterhout, R., Van Dien, S. J., and Rabinowitz, J. D. 2009 Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5: 593−599.

Keywords: Ribozyme, Chelated Mg2+, In vivo-like condition