A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes - Progress in Molecular Biology and Translational Science - page 96

Figure 3.3 The sequence and secondary structure of the VS ribozyme. 19 The proposed
secondary structure comprises seven helical segments (labeled I through VII) connected
by three three-way helical junctions. Helix I can be considered as the substrate and it can
be physically disconnected from a -acting ribozyme consisting of helices II through

VI. Cleavage and ligation reactions take place within the internal loop of helix I at the
position indicated by an arrow. Two critical nucleotides for catalytic activity are A756
(within the A730 loop of helix VI), 20,21 and G638 within the internal loop of helix I. 22

trans

junction. FRET studies with this species showed that the junction folds by
coaxial stacking of helices A on D and B on C in the presence of metal ions, 11
reminiscent of the folding of the four-way DNA junction. 27 Indeed, the
four-way junction in isolation (i.e., with the loops removed) predominantly
folds into the same stacking conformer as the complete ribozyme, so that the
junction predisposes the ribozyme to fold correctly. 28 These studies pro-
vided the first physical evidence for loop-loop interaction. A similar analysis
was performed using the hinged form. 29 Single-molecule FRET studies of
both the hinged 30 and four-way junction 31 forms of the ribozyme revealed
metal ion-dependent dynamics, with repeated docking and undocking of
the loops. The folding of the four-way junction form occurs in two stages,
with a fast ( 100 s 1 ) scissor-like rotation of the junction that presents the

loops to each other, and a slower ( 3s 1 ) docking process. 31 The rate of

docking in this form is about 500 times faster than that measured in the
hinged form. 30 Therefore, folding is not normally rate limiting in the junc-
tion form of the ribozyme. The four-way junction is not required for
catalytic activity, but it acts as an auxiliary element that assists the folding
of the ribozyme under physiological conditions 12,13,28

similar to the loops

of the hammerhead ribozyme. 32,33