(Received for publication, January 17, 1996)
From the
The RNA 5`-triphosphatase, nucleoside triphosphate
phosphohydrolase, and guanylyltransferase activities of the vaccinia
virus mRNA capping enzyme were previously localized to an
NH-terminal 60-kDa domain of the D1R subunit. Measurement
of the relative ATPase and guanylyltransferase activities remaining in
D1R carboxyl-terminal deletion variants expressed in Escherichia
coli BL21(DE3)plysS localizes the carboxyl terminus of the active
domain to between amino acids 520 and 545. Failure to obtain a deletion
mutant with the loss of one activity indicates that the catalysis of
both reactions requires a common domain structure. Based on these
results, a truncated D1R protein terminating at amino acid 545 was
expressed in E. coli and purified to homogeneity.
D1R
was found to be kinetically equivalent to the
holoenzyme in regard to ATPase, RNA 5`-triphosphatase, and
guanylyltransferase activities. Measurement of RNA binding by mobility
shift and UV photo-cross-linking analyses also demonstrates the ability
of this domain to bind RNA independent of the methyltransferase domain,
comprised of the carboxyl terminus of D1R from amino acids
498-844 and the entire D12L subunit. RNA binding to
D1R
is substantially weaker than binding to
either the methyltransferase domain or the holoenzyme. Binding is
inhibited by 5`-OH RNA and to a lesser extent by DNA oligonucleotides
in a concentration dependent manner which correlates with the
inhibition of RNA 5`-triphosphatase activity by these same
oligonucleotides. We conclude that D1R
represents
a functionally independent domain of the mRNA capping enzyme, fully
competent in substrate binding and catalysis at both the triphosphatase
and guanylyltransferase active sites.
Vaccinia virus, a member of the Poxviridae family, is a double-stranded DNA containing virus that carries out its replication entirely within the cytoplasm of the infected host cell (for a review, see (1) ). This life cycle necessitates that most enzymes required for gene expression and DNA replication are encoded by the virus. Many of these enzymes are packaged into the virion and are active in the expression of early viral genes, immediately upon infection. These enzymes include a multisubunit RNA polymerase(2, 3) , early gene transcription initiation(4, 5) , and termination factors(6) , an mRNA capping enzyme (7, 8, 9) and a poly(A) polymerase(10, 11) .
Capping of vaccinia virus mRNA
proceeds co-transcriptionally via a sequential multistep
pathway(7) . ()
are carried out by two vaccinia enzymes present in the virion core. The heterodimeric mRNA capping enzyme catalyzes the first three reactions of this pathway: RNA 5`-triphosphatase(12, 13) , guanylyltransferase(7, 8) , and (guanine-7-)methyltransferase (7, 8, 9) . The product of these reactions is the covalent modification of the mRNA 5` terminus referred to as the cap 0 structure. Formation of cap I, the product of , requires an mRNA (nucleoside-2`-O-)methyltransferase activity encoded by a separate viral enzyme(14) .
The vaccinia virus mRNA capping
enzyme is comprised of two subunits of 97 and 33-kDa molecular
mass(7) , the products of genes D1R (15) and
D12L(16) , respectively. In addition to catalyzing the
reactions of cap 0 formation, the enzyme also possesses a general
nucleoside triphosphate phosphohydrolase activity (12, 28, 37) and is required for both the
termination of early gene transcription (6) and the initiation
of intermediate gene expression(17) . Both the intact enzyme (18, 20, 23) and the methyltransferase
subdomain (24, 40) have been expressed in Escherichia coli, allowing a preliminary localization of the
enzyme active sites. The RNA 5`-triphosphatase, nucleoside triphosphate
phosphohydrolase, and guanylyltransferase activities map to the
NH-terminal 60-kDa domain of the D1R
subunit(19, 21, 23) , while the
(guanine-7-)methyltransferase activity resides in a domain comprised of
the carboxyl terminus of D1R from amino acids 545-844 together
with D12L(22, 23, 40) . Although the AdoMet
and GTP binding sites have been localized to two regions in the
carboxyl terminus of D1R(25, 26) , full
methyltransferase activity requires the association of the D12L
subunit(23, 40) . The catalytic independence of the
methyltransferase domain has been demonstrated(24) .
The
amino-terminal 60-kDa fragment of the D1R subunit appears to be
sufficient for both guanylyltransferase and phosphohydrolase
activities. This portion of D1R contains the lysine residue at position
260 (27, 38) required for the formation of the
covalent enzyme GMP intermediate of the guanylyltransferase
reaction(29, 30, 41) . The question arises
whether the phosphohydrolase and guanylyltransferase activities in the
60-kDa NH
-terminal D1R portion of the capping enzyme may be
functionally separable into two independent domains. The domain
structure of the vaccinia virus enzyme is analogous to the capping
enzymes purified from rat liver (42) and brine shrimp (43) in which a single protein possesses both
guanylyltransferase and RNA 5`-triphosphatase activities. In the latter
enzyme, the guanylyltransferase and RNA 5`-triphosphatase activities
have been fractionated by limited tryptic digestion into 44- and 20-kDa
proteolysis products, respectively, separable by gel filtration
chromatography(43) . Full capping activity, i.e. the
ability to employ triphosphate-ended RNA as a substrate for
guanylylation, could be reconstituted in vitro by combining
the two functional subdomains. In the yeast mRNA capping enzyme, the
separation of the RNA 5`-triphosphatase and guanylyltransferase active
site is more distinct as the two activities are present in separate
subunits, encoded by different genes(31, 32) . These
latter two examples argue that the existence of two catalytic
subdomains in vaccinia D1R 60-kDa domain is possible.
In this
report, we describe in greater detail the structure-function
relationship of the D1R 60-kDa subdomain. Expression in E. coli of carboxyl-terminal deletions in the D1R gene demonstrates a
shared functional domain for the RNA 5`-triphosphatase and
guanylyltransferase catalytic sites. A kinetic characterization of the
purified D1R domain demonstrates a functional
equivalence to the holoenzyme in regard to RNA 5`-triphosphatase,
ATPase, and guanylyltransferase activity. This domain also possesses
the ability to bind triphosphorylated RNA independent of the
methyltransferase domain demonstrating that the binding of RNA to this
domain alone is sufficient for catalysis at the RNA 5`-triphosphatase
and guanylyltransferase active sites.
Figure 1:
Construction of the plasmid pET8c
D1R which directs the synthesis of
D1R
. A portion of the D1R gene from position
1501-1737 was amplified by PCR. To engineer a truncated D1R
protein terminating at amino acid 545, tandem stop codons were
introduced in frame beginning at position 1738. PCR fragment term 168
was subcloned into pET8c D1Nco
by a three-way
ligation, deleting the D1R coding sequence beyond nucleotide
1737.
Figure 3:
Purification of D1R. A, BL21(DE3)plysS pET8c D1R
was induced
with 200 µM IPTG under low temperature conditions as
described. 250 g of cells were harvested from large scale inductions
and the D1R
protein purified from bacterial
lysates according to the purification scheme listed. B,
SDS-PAGE analysis of chromatography fractions taken at different steps
during purification. Samples were separated by SDS-PAGE on a 12.5%
polyacrylamide gel and stained with Coomassie-Brilliant Blue. M
, Bio-Rad low molecular weight protein standards
((
1000): 97, 66, 45, 31, 21, and 14); L, lysate; S, S
; AS, 60% ammonium sulfate
precipitate; D, DEAE cellulose flow-through; HA,
heparin agarose; Q, Q Sepharose; HT, hydroxyapatite; Se, Sephacryl 100 HR.
Figure 2:
Relative ATPase and guanylyltransferase
activities in D1R carboxyl-terminal deletions. A family of plasmids
directing the synthesis of a set of nested carboxyl-terminal deletions
of D1R (D1R COO
) was constructed and expressed
in E. coli BL21(DE3)plysS. The truncated D1R proteins were
partially purified from bacterial lysates through a batch DEAE
cellulose chromatography step. The DEAE-cellulose fractions (10 µg
of total protein) were assayed for ATPase activity (B),
guanylyltransferase activity, measured here by the formation of the E
GMP intermediate (C), and D1R specific protein
levels by Western blot analysis (D). Each activity was
measured relative to the control supernatant, C, prepared from
BL21(DE3)plysS under identical conditions. A, map of D1R
carboxyl-terminal deletion mutants, indicating the positions of their
respective carboxyl termini. Also shown is lysine 260 of the
guanylyltransferase active site, required for the formation of the E
GMP intermediate.
Figure 4:
RNA 5`-triphosphatase and ATPase substrate
saturation kinetics comparing D1R to the intact
mRNA capping enzyme. Substrate saturation kinetics were measured for
both RNA 5`-triphosphatase (A) and ATPase (B)
activities, comparing the D1R
subdomain (
)
and the holoenzyme (
). The data presented represents an average
of three different experiments, each completed in duplicate. A, RNA 5`-triphosphatase velocities were measured in a
10-µl reaction volume for 2 min at 37 °C, quenched with EDTA
and assayed for the release of
P
by thin layer
chromatography on PEI-cellulose plates. B, ATPase velocities
were measured in a similar radiochemical assay with the following
modifications: each assay was carried out in a 20-µl reaction
volume for 5 min at 37 °C, quenched by the addition of EDTA, and
assayed for the release of radiolabeled phosphate as described for the
RNA 5`-triphosphatase.
Figure 5:
Comparison of D1R and holoenzyme guanylyltransferase kinetics. Guanylyltransferase
kinetics, including both E
GMP formation (A) and
the transguanylylation of 23-mer RNA (B-D) were
determined for D1R
(
) and the full-sized
mRNA capping enzyme (
). A, E
GMP reactions
were carried out for 60 s on ice in the presence of varying amounts of
enzyme, quenched with 2
SDS sample buffer, boiled for 5 min,
and the covalently modified enzyme separated by SDS-PAGE on 10%
polyacrylamide gels. The stoichiometry of E
GMP formation
was determined by excising the radiolabeled protein from the dried gel
and quantifying by Cerenkov counting. B, 5 µM triphosphorylated 23-mer RNA was incubated with varying
concentrations of enzyme in the presence of 100 µM GTP and
2 µCi [
-
P]GTP (800 Ci/mmol) in a 10
µl reaction volume. Reactions were carried out for 4 min at 37
°C, quenched with 2
RNA sample buffer and the guanylylated
RNA resolved by electrophoresis on 10% polyacrylamide gels containing 8 M urea. Moles of guanylylated RNA were determined by Cerenkov
counting of radiolabeled RNA excised from the dried gel. C and D, comparative guanylyltransferase substrate saturation
kinetics. Guanylyltransferase velocities were measured as described in B except either the RNA concentration was varied and the
concentration of GTP held constant at 100 µM (C)
or the GTP concentration was varied and the RNA concentration held
constant at 5 µM (D).
Figure 6:
RNA binding to mRNA capping enzyme and
its functional subdomains. The ability of the mRNA capping enzyme,
D1R, or the methyltransferase domain to bind
triphosphorylated 23-mer RNA was measured by a gel mobility shift
assay. Varying amounts of capping enzyme (A),
methyltransferase domain (B), or D1R
(C), were preincubated with 20 fmol of
[
-
P]UMP labeled RNA 23-mer (
200
cpm/fmol) for 15 min on ice. RNA-protein complexes were resolved by
native gel electrophoresis on 6% polyacrylamide gels at 4 °C under
low salt conditions. Gels were dried and visualized by autoradiography (A-C). D, the total bound RNA was calculated as
a percentage of input RNA quantified by scintillation counting of both
the shifted and free (unshifted) species excised from the dried gel.
(
) capping enzyme; (
) methyltransferase domain; (
)
D1R
.
Identical RNA binding saturation results were also
obtained when RNA of a different length (e.g. a 60-mer) or RNA
possessing a diphosphorylated 5` end was employed (data not shown).
This fact held true for both the D1R and the
methyltransferase domains. These results indicate that maximal RNA
binding is achieved at modest chain length of 23 nucleotides and that
this binding does not require the terminal 5`
-phosphate, at least
when measured under the conditions of this assay. This observation
agrees with those of Luo and Shuman(36) , indicating capped
RNA, triphosphorylated RNA, and RNA lacking a phosphorylated 5` end all
interacted with the intact capping enzyme in a similar manner.
Figure 7:
Inhibition of RNA UV photo-cross-linking
and RNA 5`-triphosphatase activity by oligonucleotides. A and B, inhibition of UV photo-linkage: 30 µl of RNA
photo-cross-linking reactions were carried out in a 96-well microtiter
plate floating in an ice slurry bath and included 3 picomol of
D1R, 1 µM [
P]UMP-labeled 23-mer RNA (
100
cpm/fmol), and varying concentrations of either 5`-OH RNA or
single-stranded DNA, both 23 nucleotides in length. Following exposure
to UV light, the samples were treated with 5 µg of RNase A,
quenched with 10 µl of 4
SDS sample buffer, the samples
boiled for 5 min, and separated by SDS-PAGE. The radiolabeled enzyme
was visualized by autoradiography (A); 0*, no RNase A
treatment. B, RNA photo-linked to either subdomain was
quantified by Cerenkov counting of the radiolabeled product excised
from the dried gel: inhibition of RNA cross-linking to
D1R
by 5`-OH RNA (
) or single-stranded DNA
(
). The percent RNA cross-linked was determined relative to the
control (100%), RNA photo-linkage measured in the absence of any
competing oligonucleotides. C, inhibition of RNA
5`-triphosphatase activity by oligonucleotides, RNA 5`-triphosphatase
activity was measured in a 10-µl reaction volume containing 5
µM [
-
P]23-mer RNA, 2 mM MgCl
, 10 nM D1R
, and
varying concentrations of diphosphorylated 23-mer RNA (
), 5`-OH
23-mer RNA (
), or single-stranded DNA (
). The inclusion of
varying concentrations of unlabeled triphosphorylated RNA (
)
represents a substrate dilution effect. All velocities were measured as
a percentage of activity relative to the control (100%
activity).
The vaccinia virus mRNA capping enzyme catalyzes three sequential reactions in the mRNA cap formation pathway(7, 8, 9) . Knowledge of the number of active sites catalyzing these reactions and their physical arrangement is fundamental to our understanding the mechanism of cap formation. Any model which addresses this issue must be consistent with the known domain structure of the enzyme. The mRNA capping enzyme is comprised of two subdomains(19, 20, 21, 23) , a 60-kDa amino-terminal domain of the large D1R subunit which possesses the RNA 5`-triphosphatase, guanylyltransferase, and nucleoside triphosphate phosphohydrolase active sites(20, 23) , and a separate heterodimeric domain comprised of the carboxyl-terminal portion of D1R from amino acids 545-844 together with the small (D12L) subunit, possessing the (guanine-7-)methyltransferase active site(23, 24, 40) . Therefore, a simple model predicting a single active site for concerted catalysis is impossible; at least two active sites must exist.
We favor a model depicting
three separate active sites, one for each step in the cap formation
pathway. The three site model requires that the RNA 5`-triphosphatase
and guanylyltransferase activities are catalyzed at separate locations,
raising the possibility that the first two active sites may reside in
two independent domains. As a first test of this hypothesis,
carboxyl-terminal deletions in the D1R gene were constructed, expressed
in E. coli, and ATPase and guanylyltransferase activities
measured. The results of these measurements confirm that the D1R 60K
subdomain is the minimal functional unit for both activities and
delimit the carboxyl terminus of this active domain to between amino
acids 520 and 545. The failure of this approach to generate deletion
mutants exhibiting only ATPase or guanylyltransferase activity may
indicate a close conformational linkage of the phosphohydrolase and
guanylyltransferase active sites. In a related experiment, we observed
that the amino-terminal deletion mutant D1R lacked any measurable ATPase activity or the ability to form the
covalent E
GMP intermediate (data not shown). These
results further support the assignment of the
ATPase/guanylyltransferase domain structure to include amino acids 1 to
approximately 545. Although the physical separation of the two active
sites has not yet been achieved, a kinetic argument for separate RNA
5`-triphosphatase and guanylyltransferase active sites can be made (37) .
The assignment of the RNA 5`-triphosphatase and
guanylyltransferase activities to the D1R subdomain potentially simplifies further structure-function
analyses. In order to purify enough of this domain, a truncated D1R
protein with a defined carboxyl terminus at amino acid 545 was cloned
and expressed in E. coli. Purification of soluble enzyme was
achieved by large-scale induction of B21(DE3)plysS D1R
under low temperature conditions followed by sequential
chromatographic fractionation of the lysate. The purification of
D1R
builds on the protocol for the purification
of the full-sized capping enzyme. Unlike the intact enzyme, however,
D1R
binds poorly to phosphocellulose or
hydroxyapatite. This property mimics the chromatographic behavior of
the D1R 60-kDa domain proteolysis product observed during the
purification of the co-expressed subunits(20, 23) . As
expected, ATPase and E
GMP activities co-eluted with the
D1R protein throughout purification. A yield of 25 mg of soluble D1R
protein was typically achieved from the lysis of 250 g of cells.
A
kinetic comparison of the D1R subdomain to the
full-sized capping enzyme demonstrated that the D1R
domain possessed comparable ATPase and RNA 5`-triphosphatase
substrate saturation kinetics. Likewise, this subdomain was fully
comparable to the holoenzyme in guanylyltransferase kinetics, both in
the stoichiometry of E
GMP formation and in the
transguanylylation of RNA. Since the kinetic behavior of
D1R
is equivalent to the intact capping enzyme,
the presence of the methyltransferase domain in the holoenzyme must not
influence the turnover of RNA at either the RNA 5`-triphosphatase or
guanylyltransferase active site. A corollary to this fact states that
the structural elements which specify RNA binding for the RNA
5`-triphosphatase and guanylyltransferase active sites must reside
within D1R
.
To investigate the relationship of
RNA binding to capping enzyme function, we employed mobility shift and
UV photo-cross-linking analyses as measures of RNA binding to both
subdomains and to the full-size capping enzyme. Each subdomain of the
mRNA capping enzyme binds to 23-mer triphosphorylated RNA in a manner
indicating that up to two molecules of enzyme bind per RNA molecule, at
high enzyme concentrations. The affinity of the methyltransferase
domain for RNA is nearly 8-fold greater than D1R and 3-fold weaker than the intact capping enzyme. This result
indicates that the methyltransferase domain possesses the major RNA
binding component of the holoenzyme.
Although the binding of
triphosphorylated RNA to the D1R subdomain is
apparently weaker than binding to the methyltransferase domain, it
represents catalytically productive binding at the RNA
5`-triphosphatase active center. This conclusion is based on the fact
that photo-linkage of triphosphorylated RNA is inhibited by 5`-OH RNA
and to a much lesser extent, single-stranded DNA in a concentration
dependent manner, consistent with the relative inhibition of RNA
5`-triphosphatase activity by these same oligonucleotides. The
relationship of this inhibition to the binding of RNA at the
guanylyltransferase active site is not addressed per se by
these experiments. We cannot differentiate between separate RNA binding
sites for each active site or a single site for the binding of
diphosphorylated RNA shared by the two catalytic sites.
In
considering these data in the fuller context of a single round of RNA
cap formation, it is surprising that that the binding of RNA to the
methyltransferase domain is roughly 8-fold stronger than binding to
D1R, yet the presence of this domain exerts no
influence on the RNA substrate saturation kinetics for either the RNA
5`-triphosphatase or guanylyltransferase activities. This fact suggests
that the binding of RNA to either active site in D1R
must not be a rate-limiting step in the reaction sequence.
Alternately, RNA binding at the methyltransferase active site is
unrelated to binding at either catalytic site in the
D1R
subdomain and perhaps reflects a binding
function separate from capping at the 5` terminus.
A weak but
specific association of RNA with D1R does fit
with the three active site model for mRNA cap formation. In this model,
the RNA 5` terminus is processed sequentially at each site and in a
fashion which is kinetically autonomous from the next step. It is
sensible that the nascent RNA binds to the first active site (i.e. the RNA 5`-triphosphatase active site) with the greatest
specificity but not necessarily the highest affinity. Subsequent to
catalysis, the product must move to the next active site; a site
exhibiting less specificity for the 5`- triphosphorylated RNA but a
greater affinity for RNA would promote this transfer.