Spermine switches a Neurospora VS ribozyme from slow Cis cleavage to fast trans cleavage.
In keeping with the known role of polyamines as counterions in RNA folding, we have found that concentrations of spermine as low as 1 microM facilitate first-order Cis cleavage and decrease the concentration of magnesium required for optimal cleavage of the VS ribozyme. Surprisingly, under certain experimental conditions, cleavage reactions at concentrations of spermine above about 20 microM were not first-order. At 100 microM spermine, about half of the RNA cleaved in an initial very fast burst, k >/= 5 min-1, about 100-fold faster than under our previously optimized conditions; the remainder of the RNA cleaved more slowly. The extent of the burst and the initial rate of cleavage were proportional to RNA concentration, suggesting that the fast phase was due to intermolecular trans cleavage involving two or more RNAs. This inference of trans cleavage was confirmed by demonstrating spermine-dependent trans cleavage of particular combinations of mutant RNAs that were each incapable of cis cleavage. The experimental conditions required to switch the VS ribozyme into trans cleavage mode are quite stringent. The RNA must be preincubated with an adequate concentration of spermine at very low ionic strength near neutral pH. Concentrations of buffers and salts typically used in in vitro studies of ribozymes, including those used in our previous characterization of the VS ribozyme, are sufficiently high that they prevent or reverse the trans cleaving RNA conformation. The ability to switch cleavage modes from cis to trans provides an experimental system to study different active conformations of VS RNA, as well as to investigate the functional consequences of polyamine-RNA interactions.
Olive
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JE
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Collins
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RA
D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009491:Neurospora / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D013096:Spermine / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D016337:RNA, Catalytic; D012331:RNA, Fungal; D013096:Spermine; D008274:Magnesium