CoChat
Sign Up
Cited by 15RNA and protein synthesis mechanismsDNA and Nucleic Acid ChemistryRNA Research and Splicing

The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability

Open access
Sieg, Jacob P., McKinley, Lauren N., Huot, Melanie J., Yennawar, Neela H., Bevilacqua, Philip C.·2022·Biochemistry
DOI 10.1021/acs.biochem.2c00488PMC PMC9669196
Open this paper in CoChat — free account

Abstract

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg 2+ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg 2+ 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 Mg 2+ in the biologically relevant free Mg 2+ range of ∼0.5 to 3 mM Mg 2+, using a Mg 2+ -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 Mg 2+ between weak and strong Mg 2+ –metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1021/acs.biochem.2c00488

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The LMW Mg2+ pool is dominated by ATP and other NTPs, which bind Mg2+ with relatively high affinity (Kd ~0.1-0.3 mM).

    “The LMW Mg2+ pool is dominated by ATP and other NTPs60 that bind Mg2+ with relatively high affinity (Kd values of ∼0.1–0.3 mM).43”

    From Metals in Motion: Understanding Labile Metal Pools in Bacteria (2025) · cites this paper as [43]

  • The metabolite pool is dominated by anions, with glutamate levels approaching 100 mM and substantial contributions from NTPs and other phosphorylated compounds.

    “The metabolite pool is also dominated by anions, with glutamate levels approaching 100 mM and substantial contributions from NTPs and other phosphorylated compounds.43”

    From Metals in Motion: Understanding Labile Metal Pools in Bacteria (2025) · cites this paper as [43]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 55

  1. [1]Cech TR; Steitz JA The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones. Cell 2014, 157 (1), 77– 94. 10.1016/j.cell.2014.03.008. 24679528 · DOI · Paper page
  2. [2]Ritchey LE; Su Z; Tang Y; Tack DC; Assmann SM; Bevilacqua PC Structure-Seq2: Sensitive and Accurate Genome-Wide Profiling of RNA Structure in Vivo. Nucleic Acids Res. 2017, 45 (14), e135– e135. 10.1093/nar/gkx533. 28637286 PMC5737731 · DOI
  3. [3]Zubradt M; Gupta P; Persad S; Lambowitz AM; Weissman JS; Rouskin S DMS-MaPseq for Genome-Wide or Targeted RNA Structure Probing in Vivo. Nat. Methods 2017, 14 (1), 75– 82. 10.1038/nmeth.4057. 27819661 PMC5508988 · DOI
  4. [4]Lan TCT; Allan MF; Malsick LE; Woo JZ; Zhu C; Zhang F; Khandwala S; Nyeo SSY; Sun Y; Guo JU; Bathe M; Näär A; Griffiths A; Rouskin S Secondary Structural Ensembles of the SARS-CoV-2 RNA Genome in Infected Cells. Nat. Commun 2022, 13 (1), 1128. 10.1038/s41467-022-28603-2. 35236847 PMC8891300 · DOI
  5. [5]Morandi E; van Hemert MJ; Incarnato D SHAPE-Guided RNA Structure Homology Search and Motif Discovery. Nat. Commun 2022, 13 (1), 1722. 10.1038/s41467-022-29398-y. 35361788 PMC8971488 · DOI
  6. [6]Leamy KA; Assmann SM; Mathews DH; Bevilacqua PC Bridging the Gap between in Vitro and in Vivo RNA Folding. Q. Rev. Biophys 2016, 49: e10. 10.1017/S003358351600007X. 27658939 PMC5269127 · DOI
  7. [7]Lambert D; Draper DE Effects of Osmolytes on RNA Secondary and Tertiary Structure Stabilities and RNA-Mg2+ Interactions. J. Mol. Biol 2007, 370 (5), 993– 1005. 10.1016/j.jmb.2007.03.080. 17555763 PMC1995082 · DOI
  8. [8]Pegram LM; Wendorff T; Erdmann R; Shkel I; Bellissimo D; Felitsky DJ; Record MT Why Hofmeister Effects of Many Salts Favor Protein Folding but Not DNA Helix Formation. Proc. Natl. Acad. Sci 2010, 107 (17), 7716– 7721. 10.1073/pnas.0913376107. 20385834 PMC2867913 · DOI
  9. [9]Lambert D; Draper DE Denaturation of RNA Secondary and Tertiary Structure by Urea: Simple Unfolded State Models and Free Energy Parameters Account for Measured m-Values. Biochemistry 2012, 51 (44), 9014– 9026. 10.1021/bi301103j. 23088364 PMC3505219 · DOI
  10. [10]Cheng X; Shkel IA; Molzahn C; Lambert D; Karim R; Record MT Quantifying Interactions of Nucleobase Atoms with Model Compounds for the Peptide Backbone and Glutamine and Asparagine Side Chains in Water. Biochemistry 2018, 57 (15), 2227– 2237. 10.1021/acs.biochem.8b00087. 29533642 PMC5903953 · DOI
  11. [11]Monteith WB; Cohen RD; Smith AE; Guzman-Cisneros E; Pielak GJ Quinary Structure Modulates Protein Stability in Cells. Proc. Natl. Acad. Sci 2015, 112 (6), 1739– 1742. 10.1073/pnas.1417415112. 25624496 PMC4330749 · DOI
  12. [12]Nakano S; Karimata HT; Kitagawa Y; Sugimoto N Facilitation of RNA Enzyme Activity in the Molecular Crowding Media of Cosolutes. J. Am. Chem. Soc 2009, 131 (46), 16881– 16888. 10.1021/ja9066628. 19874030 · DOI
  13. [14]Kilburn D; Roh JH; Behrouzi R; Briber RM; Woodson SA Crowders Perturb the Entropy of RNA Energy Landscapes to Favor Folding. J. Am. Chem. Soc 2013, 135 (27), 10055– 10063. 10.1021/ja4030098. 23773075 PMC3773054 · DOI
  14. [15]Lee H-T; Kilburn D; Behrouzi R; Briber RM; Woodson SA Molecular Crowding Overcomes the Destabilizing Effects of Mutations in a Bacterial Ribozyme. Nucleic Acids Res. 2015, 43 (2), 1170– 1176. 10.1093/nar/gku1335. 25541198 PMC4333387 · DOI · Paper page
  15. [16]Leamy KA; Yennawar NH; Bevilacqua PC Cooperative RNA Folding under Cellular Conditions Arises From Both Tertiary Structure Stabilization and Secondary Structure Destabilization. Biochemistry 2017, 56 (27), 3422– 3433. 10.1021/acs.biochem.7b00325. 28657303 PMC5542450 · DOI
  16. [17]Nakano S; Karimata H; Ohmichi T; Kawakami J; Sugimoto N The Effect of Molecular Crowding with Nucleotide Length and Cosolute Structure on DNA Duplex Stability. J. Am. Chem. Soc 2004, 126 (44), 14330– 14331. 10.1021/ja0463029. 15521733 · DOI
  17. [18]Ghosh S; Takahashi S; Ohyama T; Endoh T; Tateishi-Karimata H; Sugimoto N Nearest-Neighbor Parameters for Predicting DNA Duplex Stability in Diverse Molecular Crowding Conditions. Proc. Natl. Acad. Sci 2020, 117 (25), 14194– 14201. 10.1073/pnas.1920886117. 32522884 PMC7321957 · DOI
  18. [19]Adams MS; Znosko BM Thermodynamic Characterization and Nearest Neighbor Parameters for RNA Duplexes under Molecular Crowding Conditions. Nucleic Acids Res. 2019, 47 (7), 3658– 3666. 10.1093/nar/gkz019. 30698818 PMC6468164 · DOI
  19. [20]Tyrrell J; Weeks KM; Pielak GJ Challenge of Mimicking the Influences of the Cellular Environment on RNA Structure by PEG-Induced Macromolecular Crowding. Biochemistry 2015, 54 (42), 6447– 6453. 10.1021/acs.biochem.5b00767. 26430778 PMC4893815 · DOI
  20. [21]Stadmiller SS; Pielak GJ Protein-Complex Stability in Cells and In Vitro under Crowded Conditions. Curr. Opin. Struct. Biol 2021, 66, 183– 192. 10.1016/j.sbi.2020.10.024. 33285342 · DOI
  21. [22]You X; Nguyen AW; Jabaiah A; Sheff MA; Thorn KS; Daugherty PS Intracellular Protein Interaction Mapping with FRET Hybrids. Proc. Natl. Acad. Sci 2006, 103 (49), 18458– 18463. 10.1073/pnas.0605422103. 17130455 PMC1693684 · DOI
  22. [23]Phillip Y; Kiss V; Schreiber G Protein-Binding Dynamics Imaged in a Living Cell. Proc. Natl. Acad. Sci 2012, 109 (5), 1461– 1466. 10.1073/pnas.1112171109. 22307600 PMC3277120 · DOI
  23. [24]Sukenik S; Ren P; Gruebele M Weak Protein–Protein Interactions in Live Cells Are Quantified by Cell-Volume Modulation. Proc. Natl. Acad. Sci 2017, 114 (26), 6776– 6781. 10.1073/pnas.1700818114. 28607089 PMC5495242 · DOI
  24. [25]Hull CM; Bevilacqua PC Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR. Acc. Chem. Res 2016, 49 (6), 1242– 1249. 10.1021/acs.accounts.6b00151. 27269119 PMC5305810 · DOI
  25. [26]Uehata T; Takeuchi O RNA Recognition and Immunity—Innate Immune Sensing and Its Posttranscriptional Regulation Mechanisms. Cells 2020, 9 (7), 1701. 10.3390/cells9071701. PMC7407594 32708595 · DOI
  26. [27]Yamagami R; Sieg JP; Bevilacqua PC Functional Roles of Chelated Magnesium Ions in RNA Folding and Function. Biochemistry 2021, 60 (31), 2374– 2386. 10.1021/acs.biochem.1c00012. 34319696 PMC8747768 · DOI
  27. [28]Yamagami R; Bingaman JL; Frankel EA; Bevilacqua PC Cellular Conditions of Weakly Chelated Magnesium Ions Strongly Promote RNA Stability and Catalysis. Nat. Commun 2018, 9 (1), 2149. 10.1038/s41467-018-04415-1. 29858572 PMC5984629 · DOI
  28. [29]Yamagami R; Huang R; Bevilacqua PC Cellular Concentrations of Nucleotide Diphosphate-Chelated Magnesium Ions Accelerate Catalysis by RNA and DNA Enzymes. Biochemistry 2019, 58 (38), 3971– 3979. 10.1021/acs.biochem.9b00578. 31512860 PMC6771438 · DOI
  29. [30]Leamy KA; Yamagami R; Yennawar NH; Bevilacqua PC Single-Nucleotide Control of tRNA Folding Cooperativity under near-Cellular Conditions. Proc. Natl. Acad. Sci. U. S. A 2019, 116 (46), 23075– 23082. 10.1073/pnas.1913418116. 31666318 PMC6859320 · DOI
  30. [31]Bennett BD; Kimball EH; Gao M; Osterhout R; Van Dien SJ; Rabinowitz JD Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia Coli. Nat. Chem. Biol 2009, 5 (8), 593– 599. 10.1038/nchembio.186. 19561621 PMC2754216 · DOI · Paper page
  31. [32]Martell AE; Smith RM Critical Stability Constants; NewYork; London: Plenum Press, 1974; Vol. V1.
  32. [33]Martell AE; Smith RM Critical Stability Constants; New York, Plenum Press, 1974; Vol. V2.
  33. [34]Martell AE; Smith RM Critical Stability Constants; New York, Plenum Press, 1974; Vol. V3.
  34. [37]Berthon G Critical Evaluation of the Stability Constants of Metal Complexes of Amino Acids with Polar Side Chains (Technical Report). Pure Appl. Chem 1995, 67 (7), 1117– 1240. 10.1351/pac199567071117. · DOI
  35. [39]Grilley D; Soto AM; Draper DE Chapter 3 Direct Quantitation of Mg2+-RNA Interactions by Use of a Fluorescent Dye. In Methods in Enzymology; Biothermodynamics, Part A; Academic Press, 2009; Vol. 455, pp 71– 94. 10.1016/S0076-6879(08)04203-1. PMC3001043 19289203 · DOI
  36. [40]Puglisi JD; Tinoco I Absorbance Melting Curves of RNA. In Methods in Enzymology; RNA Processing Part A: General Methods; Academic Press, 1989; Vol. 180, pp 304– 325. 10.1016/0076-6879(89)80108-9. 2482421 · DOI
  37. [41]Xia T; SantaLucia J; Burkard ME; Kierzek R; Schroeder SJ; Jiao X; Cox C; Turner DH Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson−Crick Base Pairs. Biochemistry 1998, 37 (42), 14719– 14735. 10.1021/bi9809425. 9778347 · DOI · Paper page
  38. [42]Liu B; Shankar N; Turner DH Fluorescence Competition Assay Measurements of Free Energy Changes for RNA Pseudoknots. Biochemistry 2010, 49 (3), 623– 634. 10.1021/bi901541j. 19921809 PMC2808147 · DOI
  39. [43]Liu B; Diamond JM; Mathews DH; Turner DH Fluorescence Competition and Optical Melting Measurements of RNA Three-Way Multibranch Loops Provide a Revised Model for Thermodynamic Parameters. Biochemistry 2011, 50 (5), 640– 653. 10.1021/bi101470n. 21133351 PMC3032278 · DOI
  40. [44]You Y; Tataurov AV; Owczarzy R Measuring Thermodynamic Details of DNA Hybridization Using Fluorescence. Biopolymers 2011, 95 (7), 472– 486. 10.1002/bip.21615. 21384337 PMC3082624 · DOI
  41. [45]Adamala K; Szostak JW Non-Enzymatic Template-Directed RNA Synthesis inside Model Protocells. Science 2013, 342 (6162), 1098– 1100. 10.1126/science.1241888. 24288333 PMC4104020 · DOI · Paper page
  42. [46]Soukup GA; Breaker RR Relationship between Internucleotide Linkage Geometry and the Stability of RNA. RNA 1999, 5 (10), 1308– 1325. 10573122 PMC1369853
  43. [47]Mandal M; Boese B; Barrick JE; Winkler WC; Breaker RR Riboswitches Control Fundamental Biochemical Pathways in Bacillus Subtilis and Other Bacteria. Cell 2003, 113 (5), 577– 586. 10.1016/S0092-8674(03)00391-X. 12787499 · DOI
  44. [48]Batey RT; Gilbert SD; Montange RK Structure of a Natural Guanine-Responsive Riboswitch Complexed with the Metabolite Hypoxanthine. Nature 2004, 432 (7015), 411. 10.1038/nature03037. 15549109 · DOI
  45. [49]Gilbert SD; Love CE; Edwards AL; Batey RT Mutational Analysis of the Purine Riboswitch Aptamer Domain. Biochemistry 2007, 46 (46), 13297– 13309. 10.1021/bi700410g. 17960911 PMC2556308 · DOI
  46. [50]Poudyal RR; Sieg JP; Portz B; Keating CD; Bevilacqua PC RNA Sequence and Structure Control Assembly and Function of RNA Condensates. RNA 2021, 27 (12), 1589– 1601. 10.1261/rna.078875.121. 34551999 PMC8594466 · DOI
  47. [51]Knight CJ; Hub JS WAXSiS: A Web Server for the Calculation of SAXS/WAXS Curves Based on Explicit-Solvent Molecular Dynamics. Nucleic Acids Res. 2015, 43 (W1), W225– W230. 10.1093/nar/gkv309. 25855813 PMC4489308 · DOI
  48. [52]Grant TD Ab Initio Electron Density Determination Directly from Solution Scattering Data. Nat. Methods 2018, 15 (3), 191– 193. 10.1038/nmeth.4581. 29377013 · DOI
  49. [53]Chen J-H; Yajima R; Chadalavada DM; Chase E; Bevilacqua PC; Golden BL A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests Both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage. Biochemistry 2010, 49 (31), 6508– 6518. 10.1021/bi100670p. 20677830 · DOI
  50. [54]Chadalavada DM; Gratton EA; Bevilacqua PC The Human HDV-like CPEB3 Ribozyme Is Intrinsically Fast-Reacting. Biochemistry 2010, 49 (25), 5321– 5330. 10.1021/bi100434c. 20524672 PMC2890282 · DOI
  51. [55]Robertus JD; Ladner JE; Finch JT; Rhodes D; Brown RS; Clark BFC; Klug A Structure of Yeast Phenylalanine tRNA at 3 Å Resolution. Nature 1974, 250 (5467), 546– 551. 10.1038/250546a0. 4602655 · DOI
  52. [57]Klosin A; Oltsch F; Harmon T; Honigmann A; Jülicher F; Hyman AA; Zechner C Phase Separation Provides a Mechanism to Reduce Noise in Cells. Science 2020, 367 (6476), 464– 468. 10.1126/science.aav6691. 31974256 · DOI
  53. [58]Riback JA; Zhu L; Ferrolino MC; Tolbert M; Mitrea DM; Sanders DW; Wei M-T; Kriwacki RW; Brangwynne CP Composition-Dependent Thermodynamics of Intracellular Phase Separation. Nature 2020, 581 (7807), 209– 214. 10.1038/s41586-020-2256-2. 32405004 PMC7733533 · DOI
  54. [59]Mitchell D; Russell R Folding Pathways of the Tetrahymena Ribozyme. J. Mol. Biol 2014, 426 (12), 2300– 2312. 10.1016/j.jmb.2014.04.011. 24747051 PMC4177526 · DOI
  55. [60]Williams AM; Dickson T; Lagoa-Miguel C; Bevilacqua PC Biological Solution Conditions and Flanking Sequence Modulate LLPS of RNA G-Quadruplex Structures. RNA 2022, 28, 1197– 1209. 35760522 PMC9380743

Cited by

Sorted by citations

Authors
Sieg, J. P., McKinley, L. N., Huot, M. J., Yennawar, N. H., & Bevilacqua, P. C.
Affiliations
Pennsylvania State University (US)
Corresponding author
Bevilacqua, Philip C.
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2022-10-28
Volume
61
Issue
22
Pages
2579-2591
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/acs.biochem.2c00488
PMC
PMC9669196
OpenAlex
W4307582690
Cited by
15
References
2
Citations by year
2026: 2 · 2025: 3 · 2024: 5 · 2023: 5
FWCI
1.06
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institute of General Medical Sciences (R35-GM127064)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
RNA catalysis, RNA structure
MeSH terms
Escherichia coli, RNA, Thermodynamics, RNA Stability, Metabolome

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review