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demonstrated levels of activity comparable to a known HCV IRES in both
in vitro and in vivo translation assays. The amount of luciferase produced from
the ribozyme constructs was markedly higher than the levels obtained from
the HCV IRES in vitro , with the catalytically inactive mutant ribozymes
showing the highest levels of expression. This difference was much less pro-
nounced during in vivo experiments using transfected S2 cells, perhaps due to
insect-specific translation factors missing from the rabbit reticulocyte lysate
used for in vitro studies. Regardless, expression was found to vary among
ribozymes, with the D . simulans sequences producing the highest amounts
of luciferase.
In R2 ribozymes, the J1.1/4 and P4 junction contains the consensus
sequence “RUG” that would code for either a canonical methionine as
the first amino acid, or a valine residue. The lack of conservation observed
at this presumed translation start site indicates that if this location is indeed
responsible for translation initiation, then it likely does so in a noncanonical
manner that does not depend on an AUG start codon.
Within the J4/2 region of the catalytic core, a “URA” stop codon can be
found in-frame with the upstream RUG codon, and the small open-reading
frame would yield a seven amino acid peptide. This small open-
reading frame could serve to localize and prime the remainder of the mRNA
for translation by the ribosome, as little nucleotide conservation is found in
the P4 region between isolates. However, if the URA codon does not
completely halt translation, elongation would proceed through the 3 0 side
of the P2 helix and incorporate a highly conserved amino acid sequence.
With the exception of the R2 ribozyme from D . pseudoobscura , the muta-
tions found in the P2 helix are all silent with respect to amino acid sequence.
These observations thus support a model in which the ribozyme serves to
initiate translation in a noncanonical manner during which the amino acids
coded for by the P2 helix form the N -terminus of the R2 protein of the ret-
rotransposon ( Fig. 4.8 B).
Although the R2 ribozymes exhibit minimal sequence variation, their
catalytic rates are quite divergent. 126 In the HDV and CPEB3 ribozymes,
the upstream sequences have been shown to markedly decrease self-cleavage
activity if they are capable of extending the P1 helix by base pairing with
nucleotides required to form the P1.1 helix, thereby disrupting active-site
formation of the molecule. 55,56,59,98 An identical event is observed among
the R2 ribozymes. As the number of alternate base pairs that the upstream
sequence can form is increased from zero to three, the rate constants decrease
from approximately 80 to 0.30 per hour, respectively. 126
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