an RdRp, is a
viral polymerase that catalyzes both replication and transcription in
Φ6. Φ6pol can use either ssRNA or
dsRNA as a template, producing dsRNA in either case.
single-stranded region is transcribed from dsRNA during transcription
in the 3' to 5' direction (Solgado 2004). Assembly of the initiation
de novo. Because synthesis can
occur without the use of a primer, viral RNA can be synthesized more
readily in host organisms, which do not constitutively express viral
is a bacteriophage Φ6 enzyme that synthesizes de
novo dsRNA from either single
stranded or double stranded templates
(Butcher et al. 2001). The 664-residue spherical protein resembles the
cupped right hand RNA polymerase structure, including the finger
al. 2001; Koivunen et al. 2008). NTPs are recruited
pore, which is adjacent to the RNA synthesis catalytic site. If
without the need for an additional enzyme. The coding strand is
into the template
is rich in basic
amino acid residues (Bruenn, 2003). Further,
the binding site and acts, along with other ions, to stabilize
Structure of NTP pore and active site
recruited into the enzyme through a substrate pore which
stabilizes and properly orients them prior to polymerization. At the
entrance of the
NTP tunnel (site I), basic
interact with the
phosphate groups of incoming NTPs, positioning them in the proper
orientation to polymerize (Butcher et al. 2001; Poranen et al.
The first incoming nucleotide, often CTP, interacts directly with both
the template RNA and the RdRp itself. The NTP passes beyond site I into
the subsequent binding site (site P) of the RdRp, where it stacks
In addition, the NTP polyermerizes here, base pairing with the second
of the template strand (T2). A second
NTP then base pairs at the first template residue (T1) and at the P
site (Poranen et al. 2008). This process continues until the nascent
transcript has reached a critical length. Elongation then occurs once
strand of dsRNA has displaced a C-terminal
(Poranen et al.
binds in short oligonucleotides to the RdRp in a channel known as
the template tunnel. Within this tunnel, many amino acids act to
stabilize the polymer and bind it tightly to the channel. Cytidine, the
preferred 3’ nucleotide, is bound in a pocket known as site
far past the main catalytic
site. This NTP faces the critical loop region on
and hydrogen bonds with
the main chain carbonyl group of Q629
its base hydrogen bonds
to the side chain of the
the aromatic ring of
and the ribose ring is
stabilized by hydrogen bonds
between the O2’ group and the side chain of T633
and the main chain of E634
second incoming NTP
engages in hydrophobic interactions with R291
third incoming NTP
base stacks with the previously bound NTP and hydrogen bonds with G275,
et al. 2004).
Oligonucleotide binding is illustrated in this
are both necessary as bond-mediating
to ensure φ6 RdRp function (Butcher et al. 2002). Mg2+
confers a greater degree of substrate specificity (Salgado et al.
is inhibitory at high concentrations (Butcher et al. 2002), but at low
concentrations coordinates the catalytic active site and facilitates
NTP binding (Poranen et al. 2008). One of the primary binding sites for
which is located in the
Binding induces a conformational
change in the side chain of the aspartic acid whereupon hydrogen bonds
form between the residue and the daughter sequence. The sugar of the
first nucleotide and the phosphate backbone of the second
comprise the bonds. E491
are also important binding sites for Mn2+
(Poranen et al. 2008).
forms lacking available Mn2+
ions, Q491 reorients to mediate a bond directly to A495 and D454, in a
similar fashion to the Mn ion coordination in the wt (Poranen et al.
S., L. Tomei, F. Rey, and R. De Francesco. 2002. Structural analysis of
the hepatitis C virus in complex with ribonucleotides. J. Virol.
J. 1991. Relationships among the positive strand and double-stranded
RNA viruses as views through their RNA-dependent RNA polymerases. Nucl.
Acids Res. 19(2):217-226.
S. J., J. M.
Grimes, E. V. Makeyev, D. H. Bamford, and D. I. Stuart. 2000. A
mechanism for initiating RNA-dependent RNA polymerization. Nature
M. R. L., L. P.
Sarin, D. H. Bamford. 2008. Structure-function insights into the
RNA-dependent RNA polymerase of the dsRNA bacteriophage φ6. In:
Double-stranded RNA Viruses: Structure and Molecular Biology,
J. T. Patton. Caister Academic Press, Norfolk, pp. 239-258.
Precise packing of the three genomic segments of the
M. M, P. S.
Salgado, M. R. L. Koivunen, S. Wright, D. H. Bamford, D. I. Stuart, and
J. M. Grimes. 2008. Structural explanation for the role of Mn2+
in the activity of φ6 RNA-dependent RNA polymerase. Nucl. Acids
P. S., E. V.
Makeyev, S. J. Butcher, D. H. Bamford, D. K. Stuart, and J. M. Grimes.
2004. The structural basis for RNA specificity and Ca2+
inhibition of an RNA-dependent RNA polymerase. Structure. 12:307-316.
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