Φ6pol,
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.
A
single-stranded region is transcribed from dsRNA during transcription
in the 3' to 5' direction (Solgado 2004). Assembly of the initiation
complex occurs
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
proteins
(Solgado 2004).
II.
General Structure
RNA-dependent
RNA polymerase
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
canonical
cupped right hand RNA polymerase structure, including the finger
, palm
, and
thumb
regions
(Butcher et
al. 2001; Koivunen et al. 2008). NTPs are recruited
to binding
sites
(Butcher
et al.
2001) within
the substrate
pore, which is adjacent to the RNA synthesis catalytic site. If
necessary, a
plough-like
structure
separates dsRNA
without the need for an additional enzyme. The coding strand is
directed
into the template
tunnel
which
is rich in basic
amino acid residues (Bruenn, 2003). Further,
Mn2+
is
present in
the binding site and acts, along with other ions, to stabilize
protein-RNA interactions.
III.
Structure of NTP pore and active site
NTPs are
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
residues K223,
R225,
R268,
and R270
interact with the
phosphate groups of incoming NTPs, positioning them in the proper
orientation to polymerize (Butcher et al. 2001; Poranen et al.
2008).
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
against Y630
.
In addition, the NTP polyermerizes here, base pairing with the second
residue
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
the daughter
strand of dsRNA has displaced a C-terminal
subdomain
(Poranen et al.
2008).
IV.
RNA Binding
RNA
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
S,
far past the main catalytic
site. This NTP faces the critical loop region on
Y630-K631-W632
and hydrogen bonds with
the main chain carbonyl group of Q629
;
its base hydrogen bonds
to the side chain of the
neighboring K451
and
the aromatic ring of
Y295
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
. The
second incoming NTP
engages in hydrophobic interactions with R291
and A272
. The
third incoming NTP
base stacks with the previously bound NTP and hydrogen bonds with G275,
M273,
R204,
and
K543
(Salgado
et al. 2004).
Oligonucleotide binding is illustrated in this
figure.
V.
Metal-ion interactions
Mg2+
and Mn2+
are both necessary as bond-mediating
metal ions
to ensure φ6 RdRp function (Butcher et al. 2002). Mg2+
confers a greater degree of substrate specificity (Salgado et al.
2002). Mn2+
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
Mn2+
is D454
,
which is located in the
palm domain.
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
and A495
are also important binding sites for Mn2+
(Poranen et al. 2008).
In
mutant
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.
2008).
VI.
References
Bressanelli,
S., L. Tomei, F. Rey, and R. De Francesco. 2002. Structural analysis of
the hepatitis C virus in complex with ribonucleotides. J. Virol.
76(7):3482-2492.
Bruenn,
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.
Butcher,
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
410:235-240.
Koivunen,
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:
Segmented
Double-stranded RNA Viruses: Structure and Molecular Biology,
ed.
J. T. Patton. Caister Academic Press, Norfolk, pp. 239-258.
Mindich,
Leonard. 1999.
Precise packing of the three genomic segments of the
double-stranded-RNA bacteriophage.
Poranen,
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
Res. 36(20):6633-6644.
Salgado,
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.
Back to Top