The hairpin ribozyme comprises two formally unpaired loops
carried on two arms of a four-way helical RNA junction. Addition
of divalent metal ions brings about a conformational transition
into an antiparallel structure in which there is an intimate
association between the loops to generate the active form of
the ribozyme. In this study, we have used fluorescence resonance
energy transfer to analyze the global folding of the complete
ribozyme, and the simple four-way junction derived from it,
over a wide concentration range of divalent and monovalent metal
ions. The simple junction undergoes an ion-induced rotation
into an antiparallel form. In the presence of a constant background
concentration of sodium ions, the magnesium-ion-induced transition
is characterized by noncooperative binding with a Hill coefficient
n = 1. By contrast, the magnesium-ion-induced folding
of the complete ribozyme is more complex, involving two distinct
binding phases. The first phase occurs in the micromolar range,
and involves the cooperative binding of at least three magnesium
ions. This can also be achieved by high concentrations of sodium
ions, and is therefore likely to be due to diffuse binding of
cations at the junction and the interface of the loop–loop
interaction. The second phase occurs in the millimolar range,
and can only be induced by divalent metal ions. This transition
occurs in response to the noncooperative, site-specific binding
of magnesium ions. We observe a good correlation between the
extent of ion-induced folding and cleavage activity.