In its natural context, the hairpin ribozyme is
constructed around a four-way helical junction. This presents
the two loops that interact to form the active site on
adjacent arms, requiring rotation into an antiparallel
structure to bring them into proximity. In the present
study we have compared the folding of this form of the
ribozyme and subspecies lacking either the loops or the
helical junction using fluorescence resonance energy transfer.
The complete ribozyme as a four-way junction folds into
an antiparallel structure by the cooperative binding of
magnesium ions, requiring 20–40 μM for half-maximal
extent of folding ([Mg2+]1/2)
and a Hill coefficient n = 2. The isolated junction
(lacking the loops) also folds into a corresponding antiparallel
structure, but does so noncooperatively (n = 1) at a higher
magnesium ion concentration ([Mg2+]1/2
= 3 mM). Introduction of a G + 1A mutation into loop A
of the ribozyme results in a species with very similar
folding to the simple junction, and complete loss of ribozyme
activity. Removal of the junction from the ribozyme, replacing
it either with a strand break (serving as a hinge) or a
GC5 bulge, results in greatly impaired folding,
with [Mg2+]1/2 > 20
mM. The results indicate that the natural form of the ribozyme
undergoes ion-induced folding by the cooperative formation
of an antiparallel junction and loop–loop interaction
to generate the active form of the ribozyme. The four-way
junction thus provides a scaffold in the natural RNA that
facilitates the folding of the ribozyme into the active form.