Figure 4.
Figure 4: Mechanism of HDV replication initiation. a,
HDV-derived terminal stem-loops consisting of 5 or 6 bp enable
templated incorporation of the next nucleotide(s). b, Pure Pol
II–TFIIS complex cleaves the HDV antigenome terminal segment
and elongates the newly formed 3' end on the addition of NTP.
For cleavage, Pol II–scaffold complex (625 nM) was incubated
with TFIIS (1.25 M)
in transcription buffer for 60 min at 28 °C. For elongation
of the cleavage product, the reaction mixture was incubated with
1 mM NTPs at 28 °C for 20 min. For lane 5, the cleavage
reaction was stopped after 60 min ('Stop'). c, Difference
electron density omit map for the 6-bp HDV stem-loop bound to
the hybrid site of Pol II (calculated with protein phases only,
contoured at 3.0 ).
The disordered loop is indicated with a dashed line. The view is
as in Fig. 2a. d, Superposition of the RNA template–product
duplex in the HDV EC and the RdRP EC (Fig. 2) on the DNA–RNA
hybrid duplex in the transcription EC^7. Protein structures were
superimposed by fitting the active-site aspartate loops. e,
Model of initial interaction of the HDV antigenome terminal
segment with the Pol II–TFIIS complex. The stem-loop is placed
in accordance with the crystal structure (c, d) and the
downstream duplex in accordance with the location of the FC* RNA
3' stem. We predict that the HDV bulge passes the bridge helix
and active site, where cleavage occurs.
The above figure is reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2007,
450,
445-449)
copyright 2007.
M)
in transcription buffer for 60 min at 28 °C. For elongation
of the cleavage product, the reaction mixture was incubated with
1 mM NTPs at 28 °C for 20 min. For lane 5, the cleavage
reaction was stopped after 60 min ('Stop'). c, Difference
electron density omit map for the 6-bp HDV stem-loop bound to
the hybrid site of Pol II (calculated with protein phases only,
contoured at 3.0 
).
The disordered loop is indicated with a dashed line. The view is
as in Fig. 2a. d, Superposition of the RNA template–product
duplex in the HDV EC and the RdRP EC (Fig. 2) on the DNA–RNA
hybrid duplex in the transcription EC^7. Protein structures were
superimposed by fitting the active-site aspartate loops. e,
Model of initial interaction of the HDV antigenome terminal
segment with the Pol II–TFIIS complex. The stem-loop is placed
in accordance with the crystal structure (c, d) and the
downstream duplex in accordance with the location of the FC* RNA
3' stem. We predict that the HDV bulge passes the bridge helix
and active site, where cleavage occurs.