Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis

Abstract

Most RNA folding studies have been performed under non-physiological conditions of high concentrations (≥10 mM) of Mg2+free, while actual cellular concentrations of Mg2+free are only ~1 mM in a background of greater than 50 mM Mg2+total. To uncover cellular behavior of RNA, we devised cytoplasm mimic systems that include biological concentrations of amino acids, which weakly chelate Mg2+. Amino acid-chelated Mg2+ (aaCM) of ~15 mM dramatically increases RNA folding and prevents RNA degradation. Furthermore, aaCM enhance self-cleavage of several different ribozymes, up to 100,000-fold at Mg2+free of just 0.5 mM, indirectly through RNA compaction. Other metabolites that weakly chelate magnesium offer similar beneficial effects, which implies chelated magnesium may enhance RNA function in the cell in the same way. Overall, these results indicate that the states of Mg2+ should not be limited to free and bound only, as weakly bound Mg2+ strongly promotes RNA function under cellular conditions.

Macromolecular crowding effects on transcription and translation are regulated by free magnesium ion

“…Mechanisms of Mg 2+ ‐regulated crowding effects on transcription and translation are still unclear. Recent studies on the effect of Mg 2+ on tRNA folding might provide some clues . Strulson et al.…”

“…Mechanisms of Mg 2+ -regulated crowding effects on transcription and translation are still unclear. Recent studies on the effect of Mg 2+ on tRNA folding might provide some clues [28][29][30]. Strulson et al reported that 20% of PEG or dextran increased tRNA folding cooperativity under physiologically low concentrations of Mg 2+ (0.5-2 mM) and K + (140 mM) [28].…”

Biological Pathway Specificity in the Cell—Does Molecular Diversity Matter?

“…Given its rapid dissociation rate constant, the divalent ion therefore will quickly equilibrate between many Mg 2+ ‐binding sites in the cell (Figure ) so that a chelating detection probe is subject to competition for binding of Mg 2+ , especially from those competitors with a higher affinity for Mg 2+ or a higher concentration than the probe itself (Figure ). An example is the recent observation that amino acid chelated Mg 2+ pools are readily functional in promoting RNA folding and function . What fraction of Mg 2+ is detected as “free” will therefore be extremely sensitive to both the probe used and all diverse molecular species present in the cell at the time, as well as the molecular characteristics of their individual Mg 2+ binding sites.…”

“…An example is the recent observation that amino acid chelated Mg 2+ pools are readily functional in promoting RNA folding and function. [75] What fraction of Mg 2+ is detected as "free" will therefore be extremely sensitive to both the probe used and all diverse molecular species present in the cell at the time, as well as the molecular characteristics of their individual Mg 2+ binding sites. It is no wonder that much ambiguity arises from such measurements.…”

Bulky cations greatly increase the turnover of a native hammerhead ribozyme

“…[1][2][3][4][5][6][7][8] The catalytic reaction of ribozymes proceeds in the presence of divalent metal ions, such as Mg 2+ , and even in the presence of Mg 2+ complexes coordinated by amino acids or diphosphate molecules. 9,10 Since ribozymes can selectively cleave RNA substrates, synthetic small constructs of transacting ribozymes are useful for developing oligonucleotidebased technologies, for example, for the in vivo inhibition of gene functions and for the construction of biosensor probes. [11][12][13] To maximize applications of ribozymes for biotechnology, it is important to develop methods to enhance their catalytic performance.…”