Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Abstract

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼ 20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Keywords: biological cosolutes; folding cooperativity; macromolecular crowding; tRNA; temperature-dependent structure mapping.

FIGURE 1.

Secondary structure and first derivative melt curves of WT and MT tRNA^Phe. (A) Secondary structure of wild-type (WT) tRNA^Phe. WT tRNA^Phe was prepared by in vitro T7 transcription. (B) Secondary structure of mutationally weakened tertiary structure tRNA^Phe, referred to as MT tRNA^Phe. (Red) Nucleotides mutated to remove tertiary contacts. (C) First derivative melt curves parameteric in Mg²⁺concentration for WT tRNA^Phe (closed symbols) and MT tRNA^Phe (open symbols).

FIGURE 2.

Melts of WT tRNA^Phe in additives with increasing Mg²⁺ concentration. All melts were performed in the background of 10 mM sodium cacodylate (pH 7.0) and 140 mM KCl. (A–C, both columns) The effects of various crowding agents (20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. No additive (black), PEG4000 (blue), PEG8000 (red), Dextran10 (green), Dextran70 (purple), and Ficoll70 (cyan). (D–F, both columns) The effects of low-molecular-weight cosolutes (2 m, except PEG200, which was 20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. No additive (black), methanol (pink), PEG200 (cobalt blue), proline (orange), TMAO (light purple), and betaine (dark green). In certain panels, data were adjusted to have the same T_m as the buffer, designated as “T_Buffer,” so that the effects on cooperativity are clear.

FIGURE 3.

Max dA/dT plot of WT and MT tRNA^Phe with crowding and low-molecular-weight cosolute agents in increasing Mg²⁺ concentration. Max dA/dT values were calculated for the first (i.e., lowest temperature) unfolding transition from the first derivative melt curves for (A) WT and (B) MT. Experimental conditions and coloring of plots are identical to those in Figure 2. The black dashed line across each subset of conditions shows the max dA/dT in buffer alone for that condition. Bars that extend greater than the dashed line indicate an increase in folding cooperativity. Note the difference in the y-axis scales for panels A and B.

FIGURE 4.

Difference plots of melts of WT tRNA^Phe in additives minus in buffer alone with increasing Mg²⁺ concentration. All melts were performed in the background of 10 mM sodium cacodylate (pH 7.0) and 140 mM KCl. All data were adjusted to have the same T_m, designated as “T_Buffer.” (A–C) The effects of various crowding agents (20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. PEG4000 (blue), PEG8000 (red), Dextran10 (green), Dextran70 (purple), and Ficoll70 (cyan). (D–F) The effects of low-molecular-weight cosolutes (2 m, except PEG200, which was 20% [w/v]) also in 0, 0.5, and 2 mM Mg²⁺, respectively. Methanol (pink), PEG200 (cobalt blue), proline (orange), TMAO (light purple), and betaine (dark green). Difference plots show the effects of the additives on WT folding cooperativity.

FIGURE 5.

Perpendicular TGGE plot for WT and MT tRNA^Phe melting transitions. Electrophoresis is 13% PAGE and 1× THEN₁₀M_0.9 (pH 7.5) (= 33 mM Tris, 66 mM HEPES, 0.1 mM EDTA, 10 mM NaCl, 0.9 mM MgCl₂) and 4 M urea (to facilitate melting in the TGGE range). Baselines slant to the bottom right-hand corner owing to the temperature gradient, which was also observed in the blue tracking dyes (data not shown).

FIGURE 6.

Melts of MT tRNA^Phe in additives with increasing Mg²⁺ concentration. All melts were performed in the background of 10 mM sodium cacodylate (pH 7.0) and 140 mM KCl. (A–C) The effects of various crowding agents (20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. No additive (black), PEG4000 (blue), PEG8000 (red), Dextran10 (green), Dextran70 (purple), and Ficoll70 (cyan). (D–F) The effects of low-molecular-weight cosolutes (2 m, except PEG200, which was 20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. No additive (black), methanol (pink), PEG200 (cobalt blue), proline (orange), TMAO (light purple), and betaine (dark green). In certain panels, data were adjusted to have the same T_m as the buffer, designated as “T_Buffer,” so that the effects on cooperativity are clear.

FIGURE 7.

Difference plots of melts of MT tRNA^Phe in additives minus in buffer alone with increasing Mg²⁺ concentration. All melts were performed in the background of 10 mM sodium cacodylate (pH 7.0) and 140 mM KCl. All data were adjusted to have the same T_m, designated as “T_Buffer.” (A–C) The effects of various crowding agents (20% [w/v]) in 0, 0.5, and 2 mM Mg²⁺, respectively. PEG4000 (blue), PEG8000 (red), Dextran10 (green), Dextran70 (purple), and Ficoll70 (cyan). (D–F) The effects of low-molecular-weight cosolutes (2 m, except PEG200, which was 20% [w/v]) also in 0, 0.5, and 2 mM Mg²⁺, respectively. Methanol (pink), PEG200 (cobalt blue), proline (orange), TMAO (light purple), and betaine (dark green). Difference plots show the effects of the additives on MT folding cooperativity.

FIGURE 8.

Temperature-dependent SHAPE analysis of WT tRNA^Phe in the presence and absence of PEG8000 in 0.5 mM Mg²⁺. (A–C) SHAPE analysis of WT tRNA^Phe in buffer alone. (D–F) SHAPE analysis of WT tRNA^Phe in 20% (w/v) PEG8000. (A,D) Nucleotides 25–32 in the D stem and AC stem (secondary and tertiary structure interactions). (B,E) Nucleotides 55, 56, and 58 in the TψC loop (tertiary structure interactions). (C,F) Nucleotides 69, 71, and 72 in the acceptor stem (secondary structure interactions). For plots A–C buffer alone, each nucleotide was fit separately to a two-state folding model, since there was poor correlation in T_ms for global analysis. For plots D–F in PEG8000, all nucleotides were fit globally since there was excellent agreement of T_ms within each data set and panel.

SCHEME 1.

Summary of effects of cosolutes and crowders on thermostability and RNA folding cooperativity in the presence of eukaryotic free magnesium concentration of 0.5 mM Mg²⁺. A vertical arrow signifies an increase, and a horizontal arrow signifies no change.