A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes - Progress in Molecular Biology and Translational Science - page 98

the approximate coaxial alignment of helices IV, III, and VI. It was proposed
that the probable location of helix I was the cleft formed between helices II
and VI such that it could be connected to helix II and make a loop-loop
interaction with helix V. 40 Helix I of the substrate protrudes unhindered
from the complex and can be extended without loss of catalytic activity. 41
Both strands of helix II adjacent to the II-III-VI junction were found to be
protected against hydroxyl radical attack upon folding 42 ; a result consistent
with a close association between the substrate and helix II in this region. The
interaction might be mediated by 2 0 -hydroxyl groups on the substrate and
helix II. 43 nucleotide analog interference mapping (NAIM) experiments
revealed that the removal of 2 0 -hydroxyl groups at the junction-proximal
end of helix II was deleterious, 44 and the formation of an A-minor interac-
tion was suggested. 45
The isolated junctions are induced to fold by the addition of divalent
metal ions. The folding transitions are two-state in response to the nonco-
operative binding of counterions. 39,40 There is no evidence for a require-
ment

for site-specific binding of metal ions in the folding of the

ribozyme junctions.
In the absence of a crystal structure, we turned to small-angle X-ray scat-
tering (SAXS) in solution to study the component junctions of the trans -
acting form (helices II-VI) and the complete ribozyme comprising helices
I through VII. 46 Radii of gyration and maximum chord length measure-
ments confirmed the probable codirectional orientation of helices IV-III-
VI. Reconstructions based on the full scattering curves generated a family
of closely related structures. The overall electron density envelope was rather
flat, with helix-width protrusions at three corners, and a helical ridge run-
ning across the face. These features enabled us to assign the features of the
low-resolution electron density envelope to the known helical sections of
the ribozyme.
The substrate helix I has been the subject of several studies by NMR. In
one study, the upper stem-loop of helix I was truncated and terminated with
a tetraloop, 47 whereas a complete substrate strand was analyzed in another
study. 48 In both structures, the internal loop comprised two sheared G￿A
base pairs and a protonated A þ ￿C pair, the former being similar to domain
2 of the hammerhead ribozyme. 49,50 In a different study, 51 the sequence was
changed to force the proposed conformation when the terminal loop inter-
acts with that of helix V. 24 The sheared G620￿A639 pair is preserved, but
G638 interacts with both A621 and A622 in a noncoplanar manner, while
A621 is cross-strand stacked onto A639 on the minor groove side.