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.

Figure 1

Analysis of Mg²⁺ speciation in E. coli metabolite mixtures. (A) E. coli metabolome molar composition. ‘Eco80’ contains the 15 most abundant metabolites, which comprise 80% of the E. coli metabolome. ‘NTPCM’ contains the four strong Mg²⁺-chelating NTPs, and ‘WMCM’ contains 11 other weak Mg²⁺-chelating metabolites. (B-D) Effect of Mg²⁺ concentration on HQS emission without and with mixtures of metabolites that chelate Mg²⁺. Grey lines represent fits to determine the binding constant between Mg²⁺ and HQS. (E-G) Relationship between free Mg²⁺ concentration and the total Mg²⁺ concentration with mixtures of metabolites that chelate Mg²⁺. Hex bins represent a range of total and free Mg²⁺ concentrations simulated from artificial cytoplasm assuming single-site binding (colors correspond to density of simulated values in a hex bin, with yellow being most dense and purple being least dense). Triangle data points (black) are free Mg²⁺ concentrations calculated using HQS emission. Error bars represent the uncertainty in the free Mg²⁺ concentration from propagating errors in the HQS calibration curve fit. Black lines were generated using polynomial regression. The red shaded region is the biological free Mg²⁺ range of 0.5 to 3 mM. The red line is the approximate free Mg²⁺ concentration in E. coli of 2 mM. Downward red arrows represent the total Mg²⁺required to maintain 2 mM free Mg²⁺.

Figure 2

E. coli metabolite-Mg²⁺ mixtures destabilize RNA helices. (A) Layout of a fluorescence-detected binding isotherm assay in a real-time PCR machine. FAM emission is normalized to a passive ROX reference dye. (B) Fluorescence-detected binding isotherms fit to determine equilibrium constants with MeltR. Data points represent raw fluroescence data. Curves represent curve fits. Colors represent different temperatures (purple: 32.3, blue: 41.8, teal: 51.3, green: 54.6, yellow: 58.4, orange: 60.7, red: 63.1 °C). (C) Van’t Hoff relationship between the helix association equilibrium constant and temperature for helix 2:5′-CGCAUCCU-3′/5′-AGGAUGCG-3′ folding in background, Eco80, NTPCM, and WMCM. All conditions contain 2 mM free Mg²⁺, 240 mM Na⁺, and 140 mM K⁺. Points and error bars represent association constants and standard errors propagated from the fit MeltR. Lines represent fits to the van’t Hoff equation that MeltR used to calculate folding energies. (D) The Gibbs free energy at 37 °C (ΔG°₃₇) in Eco80, NTPCM, or WMCM compared to the ΔG°₃₇ in background for five RNA helices. Errors were propagated assuming 1.5% uncertainty in the ΔG°₃₇ (see methods for error analysis).

Figure 3

E. coli metabolite-Mg²⁺ mixtures stabilized the chemical structure of RNA. (A) ILP degradation mechanism facilitated by free Mg²⁺-OH⁻. (B) Secondary structure of the guanine riboswitch aptamer with tertiary contacts. (C) Degradation rate for the guanine riboswitch aptamer at each residue in different solution conditions. (D-F) Degradation rate in different conditions grouped by structure. Groupings were based on analysis of crystal structures (Table S7). SS: Single-stranded, the base was not participating in hydrogen bonding interactions with other residues. NC: non-canonical, the base was forming non-canonical hydrogen bonding interactions in the tertiary structure. WC: Watson-Crick, the base was in a helix composed mostly of Watson-Crick base pairs.

Figure 4

Eco80 supports CPEB3-ribozyme catalysis. (A) Secondary structure of the uncleaved CPEB3 ribozyme. (B) Fraction cleaved CPEB3 as a function of time fit to a single exponential. Four technical replicates are displayed. The labels ‘2 mM Free’ and ‘25 mM Free’ refer to the Mg²⁺ concentration. All conditions contain a background of 240 mM Na⁺ and 140 mM K⁺. Enough total Mg²⁺ was added to Eco80, NTPCM, and WMCM to maintain a 2 mM free Mg²⁺ concentration (Table 2). (C) Rate constant (k) for the CPEB3 ribozyme in different conditions. k_rel is the relative rate constant in comparison to the 2 mM free Mg²⁺ condition. (D) Composition of artificial cytoplasms comprised of 80% of yeast and mammalian iMBK metabolites, termed ‘Yeast80’ and ‘Mammal80’, respectively, compared to the composition of Eco80. Each box represents one abundant metabolite. ‘NTPCM’ represents nucleotide metabolites, and ‘WMCM’ represents metabolites which were expected to weakly chelate Mg²⁺, with K_Ds greater than 2 mM.

Figure 5

Models describing the destabilization of RNA helices and stabilization of RNA chemical structure by Eco80. (A) Semi-quantitative molecular representation of an RNA helix in Eco80. The average number of molecules (colored sphere models) in Eco80 that would occupy a sphere with a 50 Å radius were placed randomly around an 8 base-pair RNA helix using Pymol (blue cartoon, PDB 1SDR). Mg²⁺ ions are represented with teal spheres. Solvent (red wires) and K⁺ (blue spheres) where modeled using WAXSiS. (B-C) Mechanism for destabilization of helices by metabolites and stabilization of helices by Mg²⁺. Net effect of metabolite-chelated Mg²⁺ combines metabolite interactions (red, white, blue) favoring the unfolded state and Mg²⁺ interactions (green) favoring the helical state. (D-E) In-line degradation of the RNA backbone mediated by Mg²⁺ hydroxide species is inhibited by Mg²⁺ chelation.