The cellular environment stabilizes adenine riboswitch RNA structure - PubMed (original) (raw)
. 2013 Dec 3;52(48):8777-85.
doi: 10.1021/bi401207q. Epub 2013 Nov 20.
Affiliations
- PMID: 24215455
- PMCID: PMC4905562
- DOI: 10.1021/bi401207q
The cellular environment stabilizes adenine riboswitch RNA structure
Jillian Tyrrell et al. Biochemistry. 2013.
Abstract
There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg(2+), the approximate Mg(2+) concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg(2+) concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg(2+) alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.
Figures
Figure 1
Expression of the adenine aptamer domain in E. coli. (A) Structure of the aptamer-tRNA construct. The adenine ligand is shown as a large A. Numbering corresponds to the add aptamer domain. (B) Total cellular RNA 3 h after inducing aptamer expression, visualized by denaturing polyacrylamide gel electrophoresis. The 143-nt aptamer-tRNA chimera product is marked with an asterisk.
Figure 2
Free Mg2+ concentration in E. coli cells measured using the ion-selective fluorophore, mag-fura-2. The free concentration in cells at late-log phase is 0.8 ± 0.2 mM in standard LB media at 37 °C (arrow). The external concentration in standard LB media is 0.25 mM. Error bars show the standard deviation of the mean from three trials.
Figure 3
In-cell SHAPE. Electropherograms showing SHAPE reactivity profiles for the native sequence adenine aptamer domain obtained (A) in buffer at a near-physiological Mg2+ (1 mM) and (B) in cells. (C) Processed SHAPE reactivity profiles for the adenine aptamer domain in cells (black) and in buffer at 1 mM Mg2+ (orange). (D) Difference plot comparing SHAPE reactivities in cells compared to buffer. Only statistically significant differences are shown (two-tailed Students t-test; p < 0.02, n = 3). Open box indicates that U48 is constrained in a rare hyper-reactive conformation (Figure S7 of Supporting Information); thus, a positive difference at this position reflects a higher level of structure in cells.
Figure 4
Ligand binding by the native sequence adenine aptamer in (A) cells and (B) in buffer. When present, the 2AP ligand concentration was 1 mM. (C) Difference plot showing nucleotides with significant changes (two-tailed Students t-test; p < 0.02, n = 3) in reactivity upon ligand binding in-cells (black) and in buffer (orange).
Figure 5
SHAPE reactivity profiles for the U74G non-binding mutant (A) in the absence (gray) and presence (blue) of ligand in cells and (B) in the absence of ligand in cells (gray) and in buffer (red). (C) Significant reactivity differences in cells compared to buffer with 1 mM Mg2+ for the wild-type (solid black bars) and U74G mutant (open gray bars) (two-tailed Students t-test; p < 0.02, n=3).
Figure 6
Correlation between in-cell and in vitro SHAPE reactivities for the ligand-free aptamer domain as a function of Mg2+ concentration. Data shown are from single-stranded and terminal base-paired nucleotides (A21-U25, U31-U39, A45-C54, G59-C67, and G72-U75) because these nucleotides show the largest structurally diagnostic changes. This correlation represents an upper limit. Inclusion of helix nucleotides, most of which have larger percent errors, results in an even poorer correlation (Figure S11 of Supporting Information). The open triangle represents the correlation between in cells and in vitro reactivities for the ligand-bound aptamer domain in 1 mM Mg2+. Inset: In vitro reactivities at 1 mM Mg2+ plotted against in-cell reactivities in the absence of ligand.
Figure 7
Nucleotide-resolution effects of the intracellular environment on adenine riboswitch aptamer domain RNA structure superimposed on the accepted ligand-bound structure (PDB ID 1y26) (A) The in-cell structure with no added ligand is defined as the reference state. Significant increases and decreases in nucleotide-resolution SHAPE reactivities relative to the in-cell RNA are emphasized in red and blue, respectively. Comparisons of the in-cell state with the aptamer RNA (B) in buffer at 1 mM Mg2+, (C) in cells with added 2AP ligand, and (D) in buffer at 30 mM Mg2+. The 2AP ligand, present in panel C, is shown in black. The inverted blue triangle in panel C indicates that U48 is constrained in a hyper-reactive conformation (Figure S7 of Supporting Information); thus, a higher (red) reactivity at this position reflects increased (blue) structure in cells.
References
- Zimmerman SB, Trach SO. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 1991;222:599–620. - PubMed
- Miyamoto S, Kashiwagi K, Ito K, Watanabe S, Igarashi K. Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli. Arch. Biochem. Biophys. 1993;300:63–68. - PubMed
- Wang Y, Sarkar M, Smith AE, Krois AS, Pielak GJ. Macromolecular crowding and protein stability. J. Am. Chem. Soc. 2012;134:16614–16618. - PubMed
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