HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 145

retrotransposition cycle. The mechanism of transposition begins with
cotranscriptional processing of the element from the surrounding rRNA
followed by endonuclease-catalyzed cleavage at the insertion site of the host
DNA. Target-primed reverse transcription and second-strand synthesis
reconstruct the retrotransposon DNA for insertion at the new locus. 123,124
Until recently, the factor responsible for the cotranscriptional processing
of the element was unknown, even though the processing was discovered
over 25 years ago. 125 Recently, it has been confirmed that this event is
due to the action of a self-cleaving ribozyme of an HDV-like motif
( Fig. 4.8 ). 4 Rapid cleavage of the R2 sequence from the surrounding tran-
script was observed in vitro , indicating that the event is autocatalytic in nature
rather than dependent on additional cellular factors. Subsequent work per-
formed on truncated versions of the R2 transposon demonstrated that an
approximately 200 nt region at the start of the sequence was essential to this
process. When this region was fitted to various models of ribozyme second-
ary structures, it was found to readily assume a drz-Agam-2-like structure.
This similarity between the R2 and drz-Agam-2 family of sequences also
renders the R2 ribozymes readily identifiable in sequence- and structure-
based searches using the drz-Agam-2 core. 126
Sequence analysis of the ribozyme-containing regions of R2 elements
revealed that areas of conservation among isolates map to the conserved core
portions of the ribozyme such as the L3 and J4/2 segments, whereas diver-
gent sequences are found in the variable portions of the ribozymes, and
covariation is present between nucleotides that would form the base-paired
helices in the folded RNA structure. Thus, a consensus sequence can be
constructed in which the P3, P1.1, L3, and J4/2 portions of the ribozymes
are identical, the J1/2 region varies in both length and composition, and the
P1 and P4 helices contain mutations that maintain base pairing ( Fig 4.8 ). In
R2 ribozymes isolated from D. melanogaster , D. sechellia , D . simulans , and
D . ananassae , the P2 helix is invariant, although several nucleotides are
covaried in isolates from D . falleni and D . pseudoobscura .
The low sequence diversity in the P2 and P3 helices among most R2
ribozymes is somewhat surprising. For the P3 helix, this may be attributable
to its proximity to the active site. In all families of HDV-like ribozymes,
including the viral sequences, the nucleotide composition of this helix con-
sists of an A-U base pair followed by two C-G base pairs, or simply three
C-G base pairs. 2,3,34 In the genomic HDV ribozyme, only the cytosine of
the middle C-G base pair (C18-G29) forms any contacts with the remainder
of the structure, forming a hydrogen bond between the C18 O2 position