title: Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions.
abstract: 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.
authors.affiliation: Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Strulson
authors.forename: Neela H
authors.initials: NH
authors.lastname: Yennawar
authors.forename: Robert P
authors.initials: RP
authors.lastname: Rambo
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bevilacqua
mesh_terms: D003698:Hepatitis Delta Virus / Q000235:genetics*; D006801:Humans; D008274:Magnesium / Q000008:administration & dosage / Q000494:pharmacology; D008958:Models, Molecular; D008970:Molecular Weight; D059370:RNA Folding / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016601:RNA-Binding Proteins; D053838:Scattering, Small Angle; D014508:Urea / Q000494:pharmacology; D014961:X-Ray Diffraction
chemical_list: C513866:CPEB3 protein, human; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D014508:Urea; D008274:Magnesium