A yeast-like mRNA capping apparatus in
C. Kiong Ho and Stewart Shuman*
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021
Communicated by Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY, December 30, 2000 (received for review December 21, 2000)
Analysis of the mRNA capping apparatus of the malaria parasite
Plasmodium falciparum illuminates an evolutionary connection to
fungi rather than metazoans. We show that P. falciparum encodes
separate RNA guanylyltransferase (Pgt1) and RNA triphosphatase
(Prt1) enzymes and that the triphosphatase component is a mem-
ber of the fungal?viral family of metal-dependent phosphohydro-
lases, which are structurally and mechanistically unrelated to the
cysteine-phosphatase-type RNA triphosphatases found in metazo-
ans and plants. These results highlight the potential for discovery
of mechanism-based antimalarial drugs designed to specifically
block the capping of Plasmodium mRNAs. A simple heuristic
scheme of eukaryotic phylogeny is suggested based on the struc-
ture and physical linkage of the triphosphatase and guanylyltrans-
ferase enzymes that catalyze cap formation.
deaths). Malaria treatment and prevention strategies have been
undermined by the spreading resistance of the Plasmodium
pathogen to erstwhile effective drugs and of the mosquito vector
to insecticides (1, 2). It is anticipated that the Plasmodium
falciparum genome project will uncover novel targets for therapy
and immunization (3). The most promising drug targets will be
those gene products or metabolic pathways that are essential for
all stages of the parasite life cycle but either absent from or
fundamentally different in the human host and the arthropod
vector. Such targets can be identified either by whole-genome
comparisons or by directed analyses of specific cellular transac-
tions. In those instances where Plasmodium differs from meta-
zoans, comparisons with other unicellular organisms may pro-
vide insights into eukaryotic phylogeny.
Here we conduct a ‘‘postgenomics’’ inquiry into the mRNA
by three enzymatic reactions: the 5?-triphosphate end of the
nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5?
triphosphatase, the diphosphate end is capped with GMP by
GTP:RNA guanylyltransferase, and the GpppN cap is methyl-
of the mRNA capping enzymes is essential for cell growth in
budding yeast. The capping apparatus differs in significant
respects in metazoans, fungi, and eukaryotic viruses (4). Mam-
mals and other metazoa encode a two-component capping
system consisting of a bifunctional triphosphatase-guanylyltrans-
ferase polypeptide and a separate methyltransferase polypep-
tide. Fungi encode a three-component system consisting of
separate triphosphatase, guanylyltransferase, and methyltrans-
ferase gene products. Viral capping systems are quite variable in
their organization; poxviruses encode a single polypeptide con-
taining all three active sites, whereas phycodnaviruses encode a
yeast-like capping apparatus in which the triphosphatase and
guanylyltransferase enzymes are encoded separately (5). The
biochemical mechanisms of the guanylyltransferase and meth-
yltransferase components of the capping apparatus are con-
served between fungi, DNA viruses, and mammals. In contrast,
the atomic structures and catalytic mechanisms of the fungal (6)
and mammalian (A. Changela, C.K.H., A. Martins, S.S., and A.
Mondragon, unpublished observations) RNA triphosphatases
alaria extracts a prodigious toll each year in human
morbidity (400 million new cases) and mortality (1 million
are completely different. The triphosphatase components of
many viral capping enzymes are mechanistically and structurally
related to the fungal proteins and not to the host cell triphos-
phatase (5, 7). Thus it has been suggested that cap formation and
RNA triphosphatase in particular are promising targets for
antifungal and antiviral drug discovery (6, 8, 9).
Little is known about the organization of the mRNA capping
apparatus in the many other branches of the eukaryotic phylo-
genetic tree. RNA guanylyltransferase has been studied in the
kinetoplastids Trypanosoma and Crithidia (10), but the triphos-
phatase and methyltransferase components have not been iden-
tified. Here we report the identification and biochemical char-
acterization of the separately encoded RNA guanylyltransferase
and RNA triphosphatase of the malaria parasite P. falciparum.
Materials and Methods
Expression and Purification of P. falciparum RNA Guanylyltransferase.
A DNA fragment containing the PGT1 ORF on chromosome 14
was amplified by PCR from total P. falciparum genomic DNA (a
gift of Derek deBruin and Jeffrey Ravetch, Rockefeller Uni-
versity) with the use of oligonucleotide primers designed to
introduce an NdeI restriction site at the predicted translation
start codon and a XhoI site 3? of the predicted stop codon.
Preliminary sequence data for P. falciparum chromosome 14 was
obtained from the Institute for Genomic Research website
and XhoI and inserted into the T7 RNA polymerase-based
expression vector pET16b to generate the plasmid pET-His-
Pgt1. The nucleotide sequence of the Plasmodium DNA insert
was determined. The predicted amino acid sequence of the
520-aa Pgt1 protein encoded by this plasmid is shown in Fig. 1.
pET-His-Pgt1 was transformed into Escherichia coli BL21-
CodonPlus(DE3). A 500-ml culture amplified from a single
transformant was grown at 37°C in LB medium containing 0.1
reached 0.5. The culture was adjusted to 2% ethanol and then
incubated at 17°C for 24 h. Cells were harvested by centrifuga-
tion, and the pellet was stored at ?80°C. All subsequent pro-
cedures were performed at 4°C. Thawed bacteria were resus-
pended in 50 ml of buffer A [50 mM Tris?HCl (pH 7.5)?0.25 M
NaCl?10% sucrose). Cell lysis was achieved by the addition of
lysozyme and Triton X-100 to final concentrations of 100 ?g?ml
and 0.1%, respectively. The lysate was sonicated to reduce
viscosity, and insoluble material was removed by centrifugation.
The soluble extract was applied to a 5-ml column of Ni-NTA-
agarose resin (Qiagen) that had been equilibrated with buffer A
containing 0.1% Triton X-100. The column was washed with 25
ml of the same buffer and then eluted stepwise with 12.5-ml
aliquots of buffer B [50 mM Tris?HCl (pH 8.0)?0.25 M NaCl?
10% glycerol?0.05% Triton X-100) containing 0.05, 0.1, 0.2, 0.5,
*To whom reprint requests should be addressed. E-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
March 13, 2001 ?
vol. 98 ?
and 1 M imidazole. The polypeptide compositions of the column
fractions were monitored by SDS-PAGE. The ?70-kDa recom-
binant Pgt1 polypeptide was recovered in the 0.1 M imidazole
fraction, which contained 5 mg of protein. The enzyme prepa-
ration was stored at ?80°C.
Guanylyltransferase Assay. Reaction mixtures (20 ?l) containing
50 mM Tris?HCl (pH 8.0), 5 mM DTT, divalent cation,
[?-32P]GTP, and Pgt1 as specified were incubated at 30°C for 10
min. The reactions were quenched with SDS, and the products
were resolved by SDS-PAGE. The Pgt1-[32P]GMP adduct was
visualized by autoradiography of the dried gel and quantitated
by scanning the gel with a FUJIX (Tokyo) PhosphorImager.
Sedimentation Analysis of Pgt1. An aliquot of the Ni-agarose
fraction of Pgt1 (50 ?g of protein) was mixed with marker
proteins catalase (50 ?g), BSA (50 ?g), and cytochrome c (50
?g), and the mixture was applied to a 4.8-ml 15–30% glycerol
DTT, and 0.05% Triton X-100. The gradient was centrifuged at
50,000 rpm for 18 h at 4°C in a Beckman SW50 rotor. Fractions
(?0.23 ml) were collected from the bottom of the tube. The
polypeptide compositions of the fractions were analyzed by
SDS-PAGE. Aliquots (2 ?l) of each fraction were assayed for
enzyme-GMP formation in a reaction mixture containing 5 mM
MnCl2and 5 ?M [?-32P]GTP.
RNA Capping by the Isolated Pgt1-GMP Intermediate. A reaction
mixture (100 ?l) containing 50 mM Tris?HCl (pH 8.0), 5 mM
DTT, 2.5 mM MgCl2, 5 ?M [?-32P]GTP, and 10 ?g of Pgt1 was
EDTA and 10% glycerol. The native Pgt1-[32P]GMP complex
was resolved from free [?-32P]GTP by gel filtration through a
1-ml column of Sephadex G-50 that had been equilibrated with
buffer C [50 mM Tris?HCl (pH 8.0)?50 mM NaCl?5 mM
DTT?10% glycerol?0.05% Triton X-100). Gel filtration was
32P elution profile was determined by Cerenkov counting of each
An aliquot (25 ?l) of the gel-filtered Pgt1-[32P]GMP complex
(recovered in the void volume of the G-50 column) was incu-
of 5? diphosphate-terminated poly(A). The reaction products
were then extracted once with phenol and once with chloroform-
isoamyl alcohol (24:1). RNA was recovered from the aqueous
phase by ethanol precipitation and resuspended in 20 ?l of 10
mM Tris?HCl (pH 8.0), 1 mM EDTA. Aliquots (4 ?l) were
digested with 5 ?g of nuclease P1 for 60 min at 37°C followed
by digestion with 1 unit of calf intestine alkaline phosphatase
for 60 min at 37°C. The digests were analyzed by TLC on
polyethyleneimine-cellulose plates developed with 0.45 M
ammonium sulfate. The radiolabeled material was visualized by
Expression and Purification of P. falciparum RNA Triphosphatase. A
DNA fragment containing the PRT1 ORF was amplified by PCR
from total P. falciparum genomic DNA with the use of oligo-
nucleotide primers designed to introduce an NcoI restriction site
at the predicted translation start codon and a BamHI site 3? of
the predicted stop codon. The PCR product was digested with
NcoI and BamHI and cloned into plasmid pYX132. The nucle-
otide sequence of the Plasmodium DNA insert was determined.
The predicted amino acid sequence of the 596-aa Prt1 protein is
shown in Fig. 1. A deletion mutant PRT1-C?140 lacking the
C-terminal 140 aa was generated by PCR amplification with a
primer designed to introduce a new stop codon and a BamHI site
immediately downstream. The C terminus of the Prt1-C?140
polypeptide is indicated by the dot above the sequence in Fig. 1.
The PCR product was digested with NcoI and BamHI, the 5?
overhangs were filled in with DNA polymerase, and the DNA
was inserted into the filled-in BamHI site of pET28-His?Smt3 (a
gift of Chris Lima, Cornell Medical College) so as to fuse the
ORF in-frame to N-terminal His6?Smt3.
pET-His?Smt3-Prt1(C?140) was transformed into E. coli
BL21-CodonPlus(DE3). A 200-ml culture amplified from a
single transformant was grown at 37°C in LB medium containing
60 ?g?ml kanamycin and 100 ?g?ml chloramphenicol until the
A600reached 0.5. The culture was adjusted to 2% ethanol and 0.4
mM isopropyl ?-D-thiogalactoside and then incubated at 17°C
for 16 h. Recombinant Prt1 was isolated from the soluble
bacterial extract by Ni-agarose chromatography as described
above for Pgt1. The recombinant His?Smt3-Prt1 polypeptide
was recovered in the 0.1 M imidazole eluate fraction. The
enzyme preparation was stored at ?80°C.
Triphosphatase Assay. Phosphohydrolase reaction mixtures (10
?l) containing 50 mM Tris?HCl (pH 7.5), 5 mM DTT, 1 mM
[?-32P]ATP or 2 ?M ?-32P-labeled poly(A), 2 mM MnCl2 or
MgCl2, and recombinant Prt1 (0.1 M imidazole eluate) as
specified were incubated for 15 min at 30°C. An aliquot (2.5 ?l)
and RNA triphosphatase Prt1. The six nucleotidyltransferase motifs in Pgt1
and phosphohydrolase motifs A, B, and C of Prt1 are highlighted in shaded
Amino acid sequences of P. falciparum RNA guanylyltransferase Pgt1
Ho and Shuman PNAS ?
March 13, 2001 ?
vol. 98 ?
no. 6 ?
of the mixture was applied to a polyethyleneimine-cellulose TLC
plate, which was developed 0.5 M LiCl, 1 M formic acid. The
radiolabeled material was visualized by autoradiography, and
32Piformation was quantitated by scanning the TLC plate with
Results and Discussion
We identified a candidate P. falciparum mRNA guanylyltrans-
ferase—a 520-aa polypeptide encoded by a continuous ORF on
chromosome 14 (Fig. 1). The signature features of mRNA
guanylyltransferases are a ping-pong reaction mechanism of
nucleotidyl transfer through a covalent enzyme-(lysyl-N)-GMP
intermediate and a set of six conserved peptide motifs (I, III,
IIIa, IV, V, and VI) involved in GTP binding and catalysis (4).
The Plasmodium guanylyltransferase (henceforth named Pgt1)
contains all six catalytic motifs in the standard order and spacing
(Fig. 2), except that the 218-aa interval between motifs IIIa and
IV of Pgt1 is exceptionally long. This segment in Pgt1 consists of
reiterative tracts of hydrophilic amino acids and has no coun-
terpart in other capping enzymes. The hydrophilic segment is
predicted, based on the crystal structure of Chlorella virus
guanylyltransferase (11), to comprise a large surface loop. All of
the amino acids within the six motifs that are essential for the
function of Saccharomyces cerevisiae RNA guanylyltransferase
Ceg1 (12) are conserved in the Plasmodium protein, as are the
residues that make direct contact with the GTP substrate in the
Chlorella virus guanylyltransferase–GTP cocrystal (11) (Fig. 2).
We produced Pgt1 in bacteria as an N-terminal His10-tagged
fusion, which allowed for rapid purification of Pgt1 based on the
affinity of the tag for immobilized nickel. The ?70-kDa Pgt1
polypeptide was adsorbed to Ni-agarose and eluted with 0.1 M
imidazole (Fig. 3A). Guanylyltransferase activity was measured
by reaction of the protein with [?-32P]GTP in the presence of a
divalent cation to form the covalent Pgt1–GMP intermediate
(Fig. 3B). Enzyme-guanylate formation was linear with respect
to Pgt1 concentration (Fig. 3C) and was strictly dependent on a
divalent cation cofactor—either manganese or magnesium (Fig.
3D). Other divalent cations—calcium, cobalt, copper and zinc—
did not support guanylyltransferase activity (data not shown).
Pgt1 formed a covalent intermediate with [?-32P]GTP but was
unable to do so with [?-32P]ATP (not shown). The rate and
extent of formation of the covalent intermediate were propor-
tional to GTP concentration and leveled off at 10 ?M GTP (Fig.
3E and data not shown). We calculated that ?20% of the input
enzyme molecules were labeled with GMP during the reaction
with 10 ?M GTP and 5 mM MnCl2. The reaction with 10 ?M
GTP displayed pseudo-first-order kinetics with an apparent rate
constant of 1.4 min?1. The native size of Pgt1 was analyzed by
glycerol gradient sedimentation with internal standards. The
guanylyltransferase activity sedimented as a single peak of 4.6 S,
which suggested that Pgt1 is a monomer in solution (Fig. 3F).
The activity profile coincided with the distribution of the Pgt1
polypeptide (not shown). That Pgt1 is a bona fide capping
enzyme was verified by isolating the Pgt1-[32P]GMP intermedi-
ate by gel filtration and demonstrating that it catalyzed transfer
of the GMP to diphosphate-terminated poly(A) to form a
GpppA cap structure that was liberated from the RNA by
digestion with nuclease P1 and was resistant to alkaline phos-
phatase (Fig. 3G).
Motif I of Pgt1 (62-KxDGxR-67) contains the lysine nucleo-
phile to which GMP becomes covalently attached during the
guanylyltransferase reaction. The position of Lys-62 relative to
the N terminus of Pgt1 is typical of the monofunctional guany-
lyltransferases of fungi and Chlorella virus. (The motif I lysine is
located at positions 70, 67, 67, and 84 in the S. cerevisiae,
Schizosaccharomyces pombe, Candida albicans, and Chlorella
virus enzymes, respectively.) The Plasmodium enzyme conspic-
uously lacks the ?200-aa N-terminal RNA triphosphatase do-
main present in metazoan and higher plant capping enzymes (4).
Metazoan RNA triphosphatases belong to a distinct branch of
the cysteine phosphatase enzyme superfamily, and they are
easily identified by their primary structure (4). We did not find
an ORF encoding a homolog of the metazoan RNA triphos-
phatase located upstream of the PGT1 gene on the P. falciparum
chromosome 14 contig (nor was such an ORF found elsewhere
in the P. falciparum genome database at National Center for
Biotechnology Information). Thus we surmise that Plasmodium
does not encode a metazoan-type mRNA capping enzyme.
The similarities between the Plasmodium and fungal guany-
lyltransferases prompted us to search for a P. falciparum
in vivo are denoted by dots. Specific contacts between amino acid side chains and the GTP substrate in the ChV capping enzyme–GTP cocrystal are indicated by
Guanylyltransferase signature motifs present in metazoan, plant, and viral capping enzymes are conserved in P. falciparum Pgt1. The amino acid
www.pnas.org?cgi?doi?10.1073?pnas.061636198 Ho and Shuman
polypeptide resembling fungal RNA triphosphatases. The S.
of metal-dependent phosphohydrolases that includes the RNA
triphosphatases encoded by other fungi (C. albicans and S.
pombe) (7, 8, 13), by algal virus PBCV-1 (5), and by several
groups of animal viruses (poxviruses, African swine fever virus,
and baculoviruses) (9, 14, 15). The yeast?viral triphosphatase
family is defined by two glutamate-rich peptide motifs (A and C)
that are essential for catalytic activity and comprise the metal-
binding site and by an intervening basic peptide motif (B) that
is implicated in binding of the 5? triphosphate moiety of the
substrate (Fig. 4). The crystal structure of S. cerevisiae Cet1
reveals that the active site is located within the hydrophilic core
of a topologically closed 8-stranded ?-barrel—the so-called
triphosphate tunnel (6). The ?-strands comprising the tunnel are
displayed over the Cet1 amino acid sequence in Fig. 4.
A PSI-BLAST search (16) initially identified a short segment of
weak similarity between Cet1 and the hypothetical P. falciparum
polypeptide PFC0985c encoded on chromosome 3 (BLAST score
42) (17). The similarity between PFC0985c and the other fungal
RNA triphosphatases was statistically significant after the first
iteration of the search (BLAST score 122). We then aligned the
Plasmodium and fungal protein sequences manually with the use
of the tertiary structure of Cet1 and known structure–activity
relationships for fungal RNA triphosphatases as a guide (6–8).
We thereby identified in the Plasmodium protein counterparts of
all eight ?-strands of the Cet1 triphosphate tunnel (Fig. 4). The
596-aa Plasmodium protein contains a 162-aa segment between
residues, that has no counterpart in other RNA triphosphatases
(Fig. 1). Reference to the Cet1 structure (6) suggests that this
segment is a surface loop emanating from the roof of the tunnel.
The instructive point is that the 12 catalytically important
hydrophilic amino acids within the tunnel that comprise the
active site of fungal RNA triphosphatases (8, 18) are conserved
in the Plasmodium protein (Fig. 4), which strongly suggests that
this protein (henceforth named Prt1) is the RNA triphosphatase
component of the Plasmodium mRNA capping apparatus.
We produced a 456-aa N-terminal domain of Prt1 in bacteria
as a His6-tagged fusion and isolated the recombinant Prt1
protein by Ni-agarose chromatography (Fig. 5A). Prt1 displayed
the signature biochemical feature of the fungal RNA triphos-
phatase family (7)—it catalyzed the hydrolysis of the ? phos-
phate of ATP in the presence of manganese (Fig. 5B). Activity
was absolutely dependent on a metal cofactor, and, as with the
fungal enzymes (7, 8, 13), magnesium was ineffective in sup-
porting ATP hydrolysis by Prt1 (Fig. 5B). ATPase activity
increased with Prt1 concentration (Fig. 5C). Prt1 also catalyzed
the magnesium-dependent hydrolysis of the ? phosphate of
triphosphate-terminated RNA (Fig. 5D).
As a member of the metal-dependent RNA triphosphatase
protein family, Prt1 is an attractive antimalarial drug target
because (i) the active site structure and catalytic mechanism of
this protein family are completely different from the RNA
triphosphatase domain of the metzoan capping enzymes and (ii)
metazoans encode no identifiable homologs of the fungal or
Plasmodium RNA triphosphatases. Thus a mechanism-based
inhibitor of Prt1 should be highly selective for the malaria
parasite and have a minimal effect on either the human host or
the mosquito vector. Given the central role of the mRNA cap in
eukaryotic gene expression, an antimalarial drug that targets
Prt1 would presumably be effective at all stages of the parasite’s
life cycle. Moreover, the structural similarity between Prt1 and
the fungal RNA triphosphatases raises the exciting possibility of
achieving antifungal and antimalarial activity with a single class
of mechanism-based inhibitors.
flow-through (FT), wash (W), and indicated imidazole eluates were analyzed by SDS-PAGE. The fixed gel was stained with Coomassie brilliant blue dye. The
positions and sizes (in kDa) of marker polypeptides are shown on the left. The Pgt1 protein is indicated by the arrowhead on the right. (B) Guanylyltransferase
mixtures contained 5 mM MgCl2, 0.17 ?M [?-32P]GTP, and Pgt1 as specified. (D) Divalent cation requirement. Reaction mixtures contained 0.17 ?M [?-32P]GTP,
200 ng of Pgt1, and either MgCl2or MnCl2as specified. (E) Kinetics. Reaction mixtures (100 ?l) containing 50 mM Tris?HCl (pH 8.0), 5 mM DTT, 5 mM MnCl2, 1
(F) Sedimentation of Pgt1 in a glycerol gradient. The guanylyltransferase activity profile is shown. The peaks of the internal marker proteins are indicated by
arrows. (G) RNA cap formation. The RNA reaction product was analyzed by TLC before and after digestion with nuclease P1 and alkaline phosphatase. The
positions of the chromatographic origin, GpppA, GTP, and Piare indicated on the right.
Characterization of P. falciparum RNA guanylyltransferase. (A) Pgt1 purification. Aliquots (15 ?l) of the soluble bacterial lysate (L), the Ni-agarose
Ho and Shuman PNAS ?
March 13, 2001 ?
vol. 98 ?
no. 6 ?
Capping enzymes are a good focal point for considering
eukaryotic evolution because the mRNA cap structure is ubiq-
uitous in eukaryotic organisms but absent from the bacterial and
archaeal kingdoms. Thus any differences in the capping appa-
ratus between taxa would reflect events that postdate the
emergence of ancestral nucleated cells. The enzymes that cata-
lyze the basic nucleic acid transactions (DNA replication, DNA
repair, RNA synthesis, and RNA processing) are generally
conserved in lower and higher eukaryotes. Yet, in the case of the
capping apparatus, we see a complete divergence of the triphos-
phatase component and the physical linkage of the triphos-
phatase and guanylyltransferase in unicellular and multicellular
This divergence suggests a heuristic scheme of eukaryotic
phylogeny based on two features of the mRNA capping appa-
ratus: the structure and mechanism of the triphosphatase com-
ponent (metal-dependent ‘‘fungal’’ type versus metal-
independent cysteine-phosphatase type) and whether the
triphosphatase is physically linked in cis to the guanylyltrans-
ferase component. By these simple criteria, relying on ‘‘black-
and-white’’ differences in the same metabolic pathway, one
arrives at relationships among taxa that are different from those
that are themselves highly conserved in all eukaryotes (19). For
example, capping-based phylogeny would place metazoans in a
common lineage with Viridiplantae (exemplified by Arabidopsis)
because all of these multicellular organisms have a cysteine-
phosphatase type RNA triphosphatase fused in cis to their
guanylyltransferase. Fungi and now Plasmodia (which are clas-
sified as Apicomplexa along with pathogenic parasites Toxo-
plasma and Cryptosporidia) fall into a different lineage distin-
guished by a ‘‘Cet1-like’’ RNA triphosphatase that is physically
separate from RNA guanylyltransferase. In contrast, the protein
sequence variation-based scheme places fungi in the same
supergroup as metazoa and puts the Apicomplexa nearer to
Assuming that multicellular organisms evolved from unicellular
ancestors, we envision an early gene rearrangement event that
sequences of the catalytic domains of fungal RNA triphosphatases of S. cerevisiae Cet1, C. albicans CaCet1, S. cerevisiae Cth1, and S. pombe Pct1. Gaps in the
degree of conservation in all five proteins are highlighted by the shaded boxes. Hydrophilic amino acids that comprise the active site within the tunnel are
denoted by dots. Conserved motifs A (?1), B (?9), and C (?11) that define the metal-dependent RNA triphosphatase family are indicated below the sequence.
The polyasparagine insert in Prt1 is omitted from the alignment and denoted by a triangle under the sequence between strands ?6 and ?7.
Structural conservation among fungal and Plasmodium RNA triphosphatases. The amino acid sequence of P. falciparum Prt1 is aligned with the
purification. Aliquots (15 ?l) of the soluble bacterial lysate (L), the Ni-agarose
flow-through (FT), wash (W), and indicated imidazole eluates were analyzed
by SDS-PAGE. The fixed gel was stained with Coomassie brilliant blue dye. (B)
Manganese-dependent NTP hydrolysis. ATPase reaction mixtures contained
0.6 ?g of Prt1 and divalent cations as specified. The reaction products were
analyzed by TLC and visualized by autoradiography. The positions of
[?-32P]ATP and32Piare indicated. (C) Prt1 titration. ATPase reaction mixtures
contained 2 mM MnCl2and Prt1 as specified. (D) RNA triphosphatase activity.
no added divalent cation, and recombinant Prt1 as specified.
Characterization of P. falciparum RNA triphosphatase. (A) Prt1
www.pnas.org?cgi?doi?10.1073?pnas.061636198 Ho and Shuman
transferredacysteine-phosphatasedomainintothesametranscrip- Download full-text
tion unit as the guanylyltransferase, leading to creation of the
in multicellular eukaryotes. The fusion presumably allowed for the
loss of a Cet1-like enzyme from the early metazoan?plant genome
or the divergence of such a protein to a point that it is no longer
independently experienced this gene fusion in distant branches of
the phylogenetic tree.
It is conceivable that, as more eukaryotic genomes are
sequenced, we will see some species containing a Cet1-like
triphosphatase fused to a guanylyltransferase, others with a
cysteine-phosphatase-type RNA triphosphatase that partici-
pates in cap formation but is physically separate from the
guanylyltransferase, and yet others that encode a novel class of
RNA triphosphatase. Nonetheless, a survey of current unicel-
lular genome databases suggests that other protozoans such as
Dictyostelium and the pathogenic parasite Trypanosoma do
indeed have ORFs encoding polypeptides that resemble fungal
RNA triphosphatases. Thus mechanism-based antimalarial
inhibitors of Plasmodium RNA triphosphatase may be effec-
tive against a battery of other unicellular parasites that cause
1. Newton, P. & White, N. (1999) Annu. Rev. Med. 50, 179–192.
2. Enserink, M. (2000) Science 287, 1956–1958.
3. Gardner, M. J. (1999) Curr. Opin. Genet. Dev. 9, 704–708.
4. Shuman, S. (2000) Prog. Nucleic Acid Res. Mol. Biol. 66, 1–40.
5. Ho, C. K., Gong, C. & Shuman, S. (2001) J. Virol. 75, 1744–1750.
6. Lima, C. D., Wang, L. K. & Shuman, S. (1999) Cell 99, 533–543.
7. Ho, C. K., Pei, Y. & Shuman, S. (1998) J. Biol. Chem. 273, 34151–34156.
8. Pei, Y., Lehman, K., Tian, L. & Shuman, S. (2000) Nucleic Acids Res. 28,
9. Ho, C. K., Martins, A. & Shuman, S. (2000) J. Virol. 74, 5486–5494.
10. Silva, E., Ullu, E., Kobayashi, R. & Tschudi, C. (1998) Mol. Cell. Biol. 18,
11. Håkansson, K., Doherty, A. J., Shuman, S. & Wigley, D. B. (1997) Cell 89,
12. Wang, S. P., Deng, L., Ho, C. K. & Shuman, S. (1997) Proc. Natl. Acad. Sci.
USA 94, 9573–9578.
13. Pei, Y., Schwer, B., Hausmann, S. & Shuman, S. (2001) Nucleic Acids Res. 29,
14. Jin, J., Dong, W. & Guarino, L. A. (1998) J. Virol. 72, 10011–10019.
15. Gross, C. H. & Shuman, S. (1998) J. Virol. 72, 10020–10028.
16. Altschul, S. F., Madden, T. L., Scha ¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W.
& Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402.
17. Bowman, S., Lawson, D., Basham, D., Brown, D., Chillingworth, T., Churcher,
C. M., Craig, A., Davies, R. M., Devlin, K., Feltwell, T., et al. (1999) Nature
(London) 400, 532–538.
18. Pei, Y., Ho, C. K., Schwer, B. & Shuman, S. (1999) J. Biol. Chem. 274, 28865–28874.
19. Baldauf, S. L., Roger, A. J., Wenk-Siefart, I. & Doolittle, W. F. (2000) Science
Ho and Shuman PNAS ?
March 13, 2001 ?
vol. 98 ?
no. 6 ?