The Huck Institutes of the Life Sciences

Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions

Strulson CA, Yennawar NH, Rambo RP, Bevilacqua PC. (2013) Biochemistry. Nov 19;52(46):8187-97.



In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that at physiological free Mg(2+) concentrations (∼0.1-0.5 mM), both crowders and cosolutes stimulate the rate of self-cleavage, up to ∼6-fold, but that in 10 mM Mg(2+) (conditions widely used for in vitro ribozyme studies) these same additives have virtually no effect on the self-cleavage rate. We further observe a dependence of the self-cleavage rate on crowder size, wherein the level of rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases at both low and high Mg(2+) concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering experiments reveal a structural basis for this stimulation in that higher-molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg(2+) that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg(2+) without a crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions.



Model of the CPEB3 ribozyme that agrees well with SAXS data. (a and c) SAXS reconstructions in (a) 0.5 mM Mg2+ and 20% PEG8000 (pink spheres) and (c) 10 mM Mg2+ without an additive (gray spheres) superimposed on the same CPEB3ribozyme model. The CPEB3 ribozyme model was constructed from the HDV ribozyme crystal structure and color-coded according to the secondary structure in Figure 1. (b and d) Experimental scattering data for the CPEB3 ribozyme in (b) 0.5 mM Mg2+ and 20% PEG8000 and (d) 10 mM Mg2+ without an additive plotted with the calculated scattering data from the native-state model. The calculated scattering profile for the CPEB3 model was generated using the FoXS server



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