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.

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

“…69 Furthermore, experiments performed in media containing the 15 most abundant metabolites of E. coli, which comprise 80% of the E. coli metabolome, demonstrated a decrease in thermodynamic stability and an increase in chemical stability (i.e., resistance to degradation) for a series of structured RNAs (the guanine aptamer, the CPEB3 ribozyme, and tRNA Phe ) of ∼2to ∼10fold relative to standard in vitro conditions, such as 2 and 25 mM free Mg 2+ . 70 Such changes could favor conformations other than P zym for the Osa 1−3 and Osa 1−8 ribozymes. It is also possible that the ribozymes may self-cleave to some extent during extraction or library preparation.…”

“…In the case of twister ribozymes, small molecules in the buffer have also been found to enhance the self-cleavage rate up to 5-fold by shuttling protons to the active site. , Such catalysis has also been shown to rescue ribozymes that would otherwise be inactive . Furthermore, experiments performed in media containing the 15 most abundant metabolites of E. coli , which comprise 80% of the E. coli metabolome, demonstrated a decrease in thermodynamic stability and an increase in chemical stability (i.e., resistance to degradation) for a series of structured RNAs (the guanine aptamer, the CPEB3 ribozyme, and tRNA Phe ) of ∼2- to ∼10-fold relative to standard in vitro conditions, such as 2 and 25 mM free Mg 2+ . Such changes could favor conformations other than P zym for the Osa 1–3 and Osa 1–8 ribozymes.…”

In vivo-like nearest neighbor parameters improve prediction of fractional RNA base-pairing in cells

“…Free energy minimization algorithms are popular, but improvements to accuracy have been modest and have stalled at ∼70% ( 13 , 16 , 17 ). Importantly, 1 M NaCl does not capture the effect of metabolites and other elements of the cellular environment ( 18 , 19 ).…”

“…Updating thermodynamic models for RNA secondary structures in cells requires the use of more biologically relevant buffer conditions. We recently presented results from a study of RNA in the Eco80 artificial cytoplasm, which contains 80% of Escherichia coli metabolites (molar basis) with biological concentrations of metal ions, including 29 mM metabolite-chelated Mg 2+ with 2 mM free Mg 2+ ( 19 ). Importantly, we determined that Eco80 provides a mixture of effects on RNA helix stability.…”

“…Importantly, we determined that Eco80 provides a mixture of effects on RNA helix stability. First, Eco80 provided destabilizing interactions with metabolites alone ( 18 , 41 ), stabilizing interactions with free Mg 2+ alone ( 42 , 43 ), and mixed destabilizing and stabilizing interactions with metabolite-chelated Mg 2+ ( 19 ). Such interactions are analogous to the quinary interactions of proteins ( 44 , 45 ).…”