Chimeras of the well-characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full-length hammerhead at equilibrium and thereby deduce the elemental cleavage (k2) and ligation (k-2) rate constants. The nine chimeras were all more active than minimal hammerheads and exhibited a very broad range of catalytic properties, with values of k2 varying by 750-fold and k-2 by 100-fold. At least two of the hammerheads exhibited an altered dependence of kobs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.
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