HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 158

(0.47 h 1 in 10 mMMg 2 þ at 37 C), as the A residue might be able to form
an alternate Watson-Crick pair with the uridine residue in the leader
sequence, extending the P1 helix and disrupting the formation of P1.1.
The self-cleavage activity of the Faecalibacterium drz-Fpra-1 ribozyme is
very temperature dependent. At 37 C, the temperature at which the bac-
teria live and grow, between 60% and 80% of the ribozyme, is found self-
cleaved in a 24 h assay ( k obs 0.47 h 1 in 10 mMMg 2 þ ). However, at room

temperature, only 20% of the ribozyme population self-cleaves within 24 h.
The cause of this phenomenon is unknown, but the fact that the ribozyme
does not self-cleave to the same endpoints at different temperatures suggests
that the sequence has been optimized to function under the conditions in
which F . prausnitzii grows.
The genomic location of drz-Fpra-1 further supports a role for the ribo-
zyme in the biochemistry of the bacterium. The ribozyme resides between
two genes: downstream of cytochrome d ubiquinol oxidase ( Cyd ) and
upstream of phosphoglucosamine mutase ( GlmM )( Fig. 4.11 ). These two
protein open-reading frames are separated by only 144 nt, indicating that
they may be part of a bi-cistronic mRNA that is processed by the ribozyme
posttranscriptionally. In the absence of additional cellular factors, the
observed slow in vitro rate of scission implies that very fewmessages are likely
to be cleaved in vivo . However, cotranscriptional self-scission has not been
measured for the ribozyme, therefore in vivo cleavage rate and extent are dif-
ficult to estimate. Additional factors that accelerate the self-cleavage activity
of drz-Fpra-1 may indeed be present, as both gene products are essential for
metabolism inside the bacteria and would likely be upregulated following
nutrient intake. 138,139 Interestingly, the bacterium contains a second copy
of the GlmM gene, along with all of the upstream drz-Fpra-1 ribozyme
except for the 5 0 side of the P1 helix. This could be due to a
retrotransposition event of the self-cleaved ribozyme-terminated GlmM
mRNA, in which the 5 0 end of the ribozyme was lost.
The drz-CIV-1 ribozyme is also found between two genes that reside
in close proximity ( Fig. 4.11 ). It maps 144 nt upstream of the viral
DNA-dependent RNA polymerase large subunit start codon, and 120 nt
downstream of the stop codon of a DNA-binding protein. Two competing
pathways have been proposed for this ribozyme. In one, the ribozyme could
serve to inactivate the translation of the large subunit of the polymerase by
cleaving the 5 0 cap of the mRNA following the initial production of the
subunit during the immediate - early stage of viral infection. 140 Alternatively,
drz-CIV-1 might

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serve to process the polymerase mRNA from the