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The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

ABSTRACT: We examined the complex network of interactions among RNA, the metabolome, and divalent Mg²⁺ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg²⁺ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivo E. coli conditions, which we term "Eco80". We empirically determined that the mixture of E. coli metabolites in Eco80 approximated single-site binding behavior toward Mg²⁺ in the biologically relevant free Mg²⁺ range of ∼0.5 to 3 mM Mg²⁺, using a Mg²⁺-sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg²⁺ between weak and strong Mg²⁺-metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

SUBMITTER: Sieg JP

PROVIDER: S-EPMC9669196 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

Sieg Jacob P JP McKinley Lauren N LN Huot Melanie J MJ Yennawar Neela H NH Bevilacqua Philip C PC

Biochemistry 20221028 22

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg2+ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg2+ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivo E. coli conditions, which ...[more]

PMID: 36306436

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Project description:Many naturally occurring RNA structures contain single mismatches, many of which occur near the ends of helices. However, previous thermodynamic studies have focused their efforts on thermodynamically characterizing centrally placed single mismatches. Additionally, algorithms currently used to predict secondary structure from sequence are based on two assumptions for predicting the stability of RNA duplexes containing this motif. It has been assumed that the thermodynamic contribution of small RNA motifs is independent of both its position in the duplex and the identity of the non-nearest neighbors. Thermodynamically characterizing single mismatches three nucleotides from both the 3' and 5' ends (i.e., off-center) of an RNA duplex and comparing these results to those of the same single mismatch-nearest neighbor combination centrally located have allowed for the investigation of these effects. The thermodynamic contributions of 13 single mismatch-nearest neighbor combinations are reported, but only nine combinations are studied at all three duplex positions and are used to determine trends and patterns. In general, the 5'- and 3'-shifted single mismatches are relatively similar, on average, and more favorable in free energy than centrally placed single mismatches. However, close examination and comparison shows there are several associated idiosyncrasies with these identified general trends. These peculiarities may be due, in part, to the identities of the single mismatch, the nearest neighbors, and the non-nearest neighbors, along with the effects of the single mismatch position in the duplex. The prediction algorithm recently proposed by Davis and Znosko [Davis, A. R., and Znosko, B. M. (2008) Biochemistry 47, 10178-10187] is used to predict the thermodynamic parameters of single mismatch contribution, and those values are compared to the measured values presented here. This comparison suggests the proposed model is a good approximation but could be improved by the addition of parameters that account for positional and/or non-nearest neighbor effects. However, more data are required to improve our understanding of these effects and to accurately account for them.

Dataset Information

The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

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The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability.

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