HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 156

In P . pacificus , the drz-Ppac-1 family of sequences can be found approx-
imately 30 times, with both the upstream and downstream regions highly
conserved. Conservation in regions flanking drz-Ppac-1, in conjunction
with the high copy number, suggests that the ribozymes represent a rare class
of retrotransposons with site-specific insertion points. 122 Analysis of
sequences downstream from the ribozymes indicates the presence of
RTE-like proteins. However, all the drz-Ppac-1 ribozymes appear inter-
genically so that it is unclear what the element might target for site-specific
insertion since. It is possible that the ribozymes are in fact not intergenic, but
appear so due to insufficient genome annotation in P . pacificus . High-
throughput transcriptome and proteome analysis for the organism was
recently completed, and the forthcoming data will perhaps answer some
of these questions. 137
Two independent isolates of drz-Ppac-1 have been tested in vitro .
Despite sharing almost identical nucleotide composition, the two ribozymes
exhibit very different kinetic properties. 3 The drz-Ppac-1-1 sequence was
found to self-cleave in 1 mM Mg 2 þ at room temperature ( k obs 1.2 h 1 ).

However, the second isolate, drz-Ppac-1-2, did not show any self-scission
at room temperature, and only residual activity in 1 mM Mg 2 þ at 37 C
( k obs

¼

0.78 h 1 ). Although flanking sequences are known to play a crucial
role in catalytic rates of HDV-like ribozymes, they are likely not contribut-
ing to the observed rate difference between the P . pacificus ribozymes
because they are nearly identical between the in vitro constructs. 56 The
drz-Ppac-1 sequences diverge primarily in their P4 helices, and two
base pairs also changed in the P1 helix between the two isolates. Although
the P4 helix is not essential for catalysis, it is expected to promote proper
folding of the ribozyme. Therefore, the difference in catalytic rates might
reflect the strength of base-pairing interactions in this helix.

¼

5.16. HDV-like ribozymes in bacteria and viruses
The drz-Frpa-1 and drz-CIV-1 from F . prausnitzii and CIV, respectively, are
currently the only two HDV-like ribozymes identified outside of eukaryotes
and the HDV genome. As the number of bacterial and viral strains and spe-
cies is far greater than those of eukaryotes, it is likely that many more HDV-
like ribozymes reside in these bastions of sequence space that must be
searched as new genomes and populations of microorganisms become avail-
able. Alternatively, direct, metagenomic searches from environmental sam-
ples using HDV-like motif descriptors could be used to uncover additional
HDV-like ribozymes.