The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation - Progress in Molecular Biology and Translational Science - page 18

compact sequences dominate the collection of all known ribozymes, the dis-
continuous format of the CLEC2 ribozymes is not without precedent. The
widespread nonanimal self-splicing group I and II introns span hundreds to
thousands of nucleotides. The CLEC2 ribozyme demonstrates that inter-
ruption of functional domains with long sequences does not impede ham-
merhead ribozyme activity. Further investigation will determine whether
these sequence intervals play a role in the regulation of ribozyme function
and gene expression.
The m CLEC2d -associated ribozyme is very similar to the well-studied
full-length Schistosome ribozyme that is active in physiological condi-
tions. 49 The catalytic core is strictly conserved with a single change in posi-
tion 7, a well-documented variable nucleotide, while the lengths and base
pairing of stem(s) I and II preserve the overall architecture of the secondary
structure observed in full-length hammerhead ribozymes. More remarkable,
the 16 nucleotides of stem II are also identical between the Schistosome
and the m CLEC2d -associated ribozymes except for a single-base deviation
in the loop region ( Fig. 1.3 ). In full-length hammerhead ribozymes, inter-
action between the stem II loop and stem(s) I bulge confers a catalytic rate
enhancement to the active site through tertiary structure changes and enables
activity in physiologically relevant conditions. Considering that nucleotides
in the stem(s) I bulge are also conserved between these two hammerhead
ribozymes from phylogenetically distant organisms, it is reasonable to expect
that CLEC2 ribozyme tertiary structure corresponds to that of the Schisto-
some hammerhead ribozyme and functions at physiological conditions with
similar kinetics. 20,41,66 Consistent with these comparisons, cell-based assays
using reporter gene constructs conjugated to CLEC2 3 0 UTRs demonstrated
that the embedded ribozymes significantly reduce expression of the upstream
gene by effectively cleaving and destabilizing the mRNA. 9,10
Additional features of the CLEC2 hammerhead ribozyme are universally
conserved among all hammerhead ribozymes from diverse origins highlight-
ing the importance of these elements to ribozyme activity in vivo . Naturally
occurring hammerheads as well as artificially selected motifs have rather var-
iable sequences in the loop and bulge regions with specific combinations
particular to different ribozyme sources. 67-69 One exception to this is the
widely conserved adenosine in the sixth position of Loop II of type I and
III ribozymes. In the full-length ribozyme tertiary structure, this base is
involved in a noncanonical interaction with the conserved uridine from
the substrate strand. 41,70 Preservation of this specific combination in the