Pharmacological targeting of mRNA cap formation for treatment of parasitic infections

Abstract

This invention provides the genes encoding the RNA triphosphatase and RNA guanylyltransferase of the malaria parasite Plasmodium falciparum and the catalytically active recombinant RNA triphosphatase and RNA guanylyltransferase enzymes. These enzymes form the basis of activity inhibition assays to identify molecules that specifically target the formation of the mRNA 5′ cap in unicellular eukaryotic parasites.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biochemical pharmacology and drug discovery. More specifically, the present invention relates to the novel mRNA capping enzymes Pgt1 and Pgt1 from Plasmodium falciparum , the agent of malaria, and methods of screening for antimalarial and antiprotozoal compounds that inhibit mRNA cap formation.

2. Description of the Related Art

Malaria extracts a prodigious toll each year in human morbidity (400 million new cases) and mortality (1 million deaths). The malaria parasite is transmitted when humans are bitten by the Anopheles mosquito. Of the four species of Plasmodium parasites that cause human malaria— Plasmodium vivax, Plasmodium malariae, Plasmodium ovale , and Plasmodium falciparum —it is P. falciparum that is principally responsible for fulminant disease and death. Malaria treatment and prevention strategies have been steadily undermined by the spreading resistance of the Plasmodium pathogen to erstwhile effective drugs and of the mosquito vector to insecticides [1]. Thus, there is an acute need for new malaria therapies.

It is anticipated that the Plasmodium falciparum genome project [2] will uncover novel targets for therapy and immunization. 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 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 transactions. In those instances where Plasmodium differs from metazoans, comparisons to other unicellular organisms may provide insights into eukaryotic phylogeny.

Processing of eukaryotic mRNA in vivo is coordinated temporally and physically with transcription. The earliest event is the modification of the 5′ terminus of the nascent transcript to form the cap structure m7GpppN. The cap is formed by three enzymatic reactions: (i) the 5′ triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA 5′ triphosphatase; (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase; and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase [3].

RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus that abrogate catalytic activity are lethal in vivo. Genetic and biochemical experiments highlight roles for the cap in protecting mRNA from untimely degradation by cellular 5′ exonucleases and in recruiting the mRNA to the ribosome during translation initiation.

The physical and functional organizations of the capping apparatus differ in significant respects in metazoans, fungi, and viruses. Mammals and other metazoa encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide and a separate methyltransferase polypeptide. Fungi encode a three-component system consisting of separate triphosphatase, guanylyltransferase, and methyltransferase gene products. Viral capping systems are quite variable in their organization; poxviruses encode a single polypeptide containing all three active sites, whereas phycodnaviruses encode a yeast-like capping apparatus in which the triphosphatase and guanylyltransferase enzymes are encoded separately [4].

The guanylyltransferase and methyltransferase components of the capping apparatus are mechanistically conserved between metazoans and budding yeast. In contrast, the structures and catalytic mechanisms of the mammalian and fungal RNA triphosphatases are completely different [5]. The triphosphatase components of many viral mRNA capping enzymes are mechanistically and structurally related to the fungal RNA triphosphatases, and not to the host cell triphosphatase [4, 6, 7]. Thus, cap formation and cap-forming enzymes, especially RNA triphosphatase, are promising targets for antifungal and antiviral drug discovery.

A plausible strategy for antimalarial drug discovery is to identify compounds that block Plasmodium-encoded capping activities without affecting the capping enzymes of the human host or the mosquito vector. For this approach to be feasible, the capping enzymes of the malaria parasite must be identified.

Little is known about the organization of the mRNA capping apparatus in the many other branches of the eukaryotic phylogenetic tree. RNA guanylyltransferase has been studied in the kinetoplastids Trypanosoma and Crithidia [8] but the triphosphatase and methyltransferase components have not been identified.

RNA Guanylyltransferase—Transfer of GMP from GTP to the 5′ diphosphate terminus of RNA occurs in a two-step reaction involving a covalent enzyme-GMP intermediate [3]. Both steps require a divalent cation cofactor.

(i) E+pppG⇄E-pG+PPi

(ii) E-pG+ppRNA⇄GpppRNA+E

The GMP is covalently linked to the enzyme through a phosphoamide (P—N) bond to the epsilon-amino group of a lysine residue within a conserved KxDG element (motif I) found in all known cellular and DNA virus-encoded capping enzymes (FIG. 1 ). Five other sequence motifs (III, IIIa, IV, V, and VI) are conserved in the same order and with similar spacing in the capping enzymes from fungi, metazoans, DNA viruses, and trypanosomes ( FIG. 1 ) [3].

H{dot over (a)}kansson et al. [9] have determined the crystal structure of the Chlorella virus guanylyltransferase in the GTP-bound state and with GMP bound covalently. The protein consist of a larger N-terminal domain (domain 1, containing motifs I, III, IIIa, and IV) and a smaller C-terminal domain (domain 2, containing motif VI) with a deep cleft between them. Motif V bridges the two domains. Motifs I, III, IIIa, IV, and V form the nucleotide binding pocket. The crystal structure reveals a large conformational change in the GTP-bound enzyme, from an “open” to a “closed” state, that brings motif VI into contact with the beta and gamma phosphates of GTP and reorients the phosphates for in-line attack by the motif I lysine.

Identification of essential amino acids has been accomplished by site-directed mutagenesis of Cegt1, the RNA guanylyltransferase of Saccharomyces cerevisiae . The guanylyltransferase activity of Cegt1p is essential for cell viability. Hence, mutational effects on Ceg1 function in vivo can be evaluated by simple exchange of mutant CEG1 alleles for the wild type gene. The effects of alanine substitutions for individual amino acids in motifs I, III, IIIa, IV, V, and VI have been examined. Sixteen residues were defined as essential (denoted by dots in FIG. 1 ) and structure-activity relationships at these positions were subsequently determined by conservative replacements [10]. Many of the essential Cegt1 side chains correspond to moieties which, in the Chlorella virus capping enzyme crystal structure, make direct contact with GTP as denoted by the arrowheads in FIG. 1 .

RNA Triphosphatase—There are at least two mechanistically and structurally distinct classes of RNA 5′ triphosphatases: (i) the divalent cation-dependent RNA triphosphatase/NTPase family (exemplified by Saccharomyces cerevisiae Cet1 and Cth1 , Candida albicans CaCet1 , Schizosaccharomyces pombe Pct1, Chlorella virus Rtp1, baculovirus LEF-4, and vaccinia virus D1), which require three conserved collinear motifs (A, B, and C) for activity [4,6,7,11-14], and (ii) the divalent cation-independent RNA triphosphatases, e.g., the metazoan cellular mRNA capping enzymes, the baculovirus phosphatase BVP, and the human enzyme PIR1, which require a HCxxxxxR(S/T) phosphate-binding motif [15-17].

Metazoan capping enzymes consist of an N-terminal RNA triphosphatase domain and a C-terminal guanylyltransferase domain. In the 497-amino acid mouse enzyme Mce1, the two catalytic domains are autonomous and nonoverlapping [15]. The metazoan RNA triphosphatases belong to a superfamily of cysteine phosphatases that includes protein tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases. The metazoan RNA triphosphatases contain a HCxxxxxR(S/T) signature motif (referred to as the P loop) that defines the cysteine phosphatase superfamily (FIG. 2 ). Metazoan RNA triphosphatases catalyze the cleavage of the γ phosphate of 5′ triphosphate RNA via a two-step pathway. First, a cysteine thiolate nucleophile of the enzyme (the conserved cysteine of the P loop) attacks the γ phosphorus to form a covalent protein-cysteinyl-S-phosphate intermediate [16] and release the diphosphate-terminated product. Then the covalent intermediate is hydrolyzed to liberate inorganic phosphate. The metazoan RNA triphosphatases do not require a metal cofactor.

Saccharomyces cerevisiae Cet1 exemplifies the class of divalent cation-dependent RNA triphosphatase enzymes, which includes the RNA triphosphatase encoded by the pathogenic fungus Candida albicans , the fission yeast Schizosaccharomyces pombe , and the RNA triphosphatase components of the capping systems of poxviruses, baculoviruses, and Chlorella virus PBCV-1. This triphosphatase family is defined by three conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids that are essential for Cet1 catalytic activity [6,12] (FIG. 3 ).

Purified recombinant Cet1 catalyzes the magnesium-dependent hydrolysis of the γ phosphate of triphosphate-terminated RNA to form a 5′ diphosphate end. Cet1 also displays a robust ATPase activity in the presence of manganese or cobalt, but magnesium, calcium, copper, and zinc are not effective cofactors for ATP hydrolysis [6]. Cet1 displays broad specificity in converting rNTPs and dNTPs to their respective diphosphates. The manganese- and cobalt-dependent NTPase activity of Cet1 resembles the manganese- or cobalt-dependent NTPase activities of the of the other members of this family, including baculovirus LEF-4 , C. albicans CaCet1 , S. cerevisiae Cth1 , S. pombe Pct1, and Chlorella virus Rtp1 [4,11-14].

Crystal Structure of Fungal RNA Triphosphatase —The biologically active triphosphatase derivative Cet1(241-539) was crystallized and its structure determined at 2.05 Å resolution [5]. Consistent with solution studies, Cet1 crystallized as a dimer. The striking feature of the tertiary structure is the formation of a topologically closed tunnel composed of 8 antiparallel β strands. The active site resides within this hydrophilic “triphosphate tunnel”. The β strands that comprise the walls of the tunnel are displayed over the Cet1 protein sequence in FIG. 3 . The interior of the tunnel contained a single sulfate ion coordinated by two arginine and two lysine side chains. Insofar as sulfate is a structural analog of phosphate, it is likely that the side chain interactions of the sulfate reflect contacts made by the enzyme with the γ phosphate of the triphosphate-terminated RNA and nucleoside triphosphate substrates.

The proteins most closely related to Cet1 at the primary structure level are CaCet1, Pct1, and Cth1. CaCet1 is the RNA triphosphatase component of the capping apparatus of Candida albicans . Pct1 is the RNA triphosphatase component of the capping apparatus of Schizosaccharomyces pombe [14]. Cth1 is a nonessential S. cerevisiae protein with divalent cation-dependent RNA triphosphatase/NTPase activity that may participate in an RNA transaction unrelated to capping [12]. The amino acid sequences of Cet1, CaCet1, Pct1, and Cth1 are aligned in FIG. 3 . The residues conserved in all four fungal enzymes are localized predominantly in the interior of the tunnel.

Cet1 triphosphatase activity is strictly dependent on a divalent cation cofactor. The hydrolysis of 5′ triphosphate RNA termini is optimal in the presence of magnesium, whereas NTP hydrolysis specifically requires manganese or cobalt. The location of a metal-binding site on the enzyme was determined by X-ray diffraction of Cet1(241-539) crystals that had been soaked in manganese chloride [5]. Manganese is coordinated with octahedral geometry to the sulfate inside the tunnel, to the side chain carboxylates of three glutamates, and to two waters. The three glutamates that comprise the metal-binding site of fungal RNA triphosphatase are located in motifs A and C, which define the metal-dependent RNA triphosphatase family. Substitution of any one of the three glutamates by alanine or glutamine inactivates Cet1. The motif A and C glutamates are also essential for the activities of vaccinia virus RNA triphosphatase, baculovirus RNA triphosphatase, C. albicans CaCet1 , S. pombe Pct1, and S. cerevisiae Cth1. Thus, it is likely that motifs A and C comprise the metal binding site in all members of this enzyme family.

The structure of Cet1(241-539) with bound sulfate and manganese is construed to reflect that of the product complex of enzyme with the hydrolyzed γ phosphate [5]. The structure suggests a catalytic mechanism whereby acidic side chains located on the floor of the tunnel coordinate an essential divalent cation that in turn coordinates the γ phosphate. The metal ion would activate the γ phosphorus for direct attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the β phosphate leaving group. Interactions between the sulfate and basic side chains located on the walls of the tunnel would contribute to the coordination of the 5′ phosphates in the ground state and the stabilization of the negative charge on the γ phosphate developed in the transition state. A key mechanistic distinction between the fungal-type RNA triphosphatases and the metazoan-type RNA triphosphatases is that the fungal-type enzymes do not form a covalent phosphoenzyme intermediate.

The prior art is deficient in the lack of methods that teach a person having ordinary skill in this art how to screen for a compound that inhibits cap formation by the enzymes of unicellular eukaryotic parasites such as Plasmodia. The prior art is also deficient in an identification and characterization of the enzymes comprising the mRNA capping apparatus of Plasmodia. In particular, the RNA triphosphatase component of the mRNA capping apparatus has not been identified and characterized in any unicellular eukaryotic parasite. The biochemical properties of an RNA triphosphatase from a unicellular eukaryotic parasite are not known. Hence, a mechanistic and structural comparison between the RNA triphosphatase of the parasite and the RNA triphosphatase of the metazoan host organism, which could underscore the potential of RNA triphosphatase as a therapeutic target for parasitic infections, is not possible. The present invention fulfills this longstanding need in the art.

SUMMARY OF THE INVENTION

The present invention facilitates the discovery of drugs that target an essential aspect of gene expression—the formation of the mRNA 5′ cap m7GpppN—in unicellular eukaryotic parasites.

The invention discloses the amino acid sequences of the Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase, which catalyze the first and second steps of mRNA cap formation, respectively. The invention also provides for expression vectors and recombinant Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase.

The invention further encompasses in vitro screening methods to identify candidate inhibitors of the catalytic activity of RNA guanylyltransferase or the RNA 5′ triphosphatase of unicellular eukaryotic parasites. These methods are simple, quantitative, and adaptable to colorimetric, spectrophotometric, or fluorescence detection assays that are suited to high-throughput screening for inhibitors of the RNA triphosphatase of Plasmodia and other unicellular eukaryotic parasites.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. 1 shows the signature motifs of cellular and viral RNA guanylyltransferases. Six collinear sequence elements, designated motifs I, III, IIIa IV, V, and VI, are present in metazoan, plant, and viral capping enzymes. The amino acid sequences are aligned for the guanylyltransferases of S. cerevisiae (Sce), S. pombe (Spo), C. albicans (Cal), Chlorella virus PBCV-1 (ChV), Caenorhabditis elegans (Cel) mouse (Mus), Drosophila melanogaster (Dme), Xenopus laevis (Xle), Arabidopsis thaliana (Ath), Trypanosoma brucei gambiense (Tbr), Crithidia fasciculata (Cfa), African swine fever virus (ASF), and AcNPV baculovirus (AcNPV). The motifs of the Plasmodium falciparum (Pfa) guanylyltransferase are listed below the other aligned sequences. The numbers of amino acid residues separating the motifs are indicated. The amino acids of the Sce enzyme that are essential for its function 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 arrowheads.

FIG. 2 shows an amino acid sequence alignment of the RNA triphosphatase domain of mammalian capping enzyme (Mcel) with other metazoan RNA capping enzymes from Xenopus laevis (Xla), Drosophila melanogaster (Dme), C elegans (Cel), and Arabidopsis thaliana (Ath) and related RNA-specific 5′ phosphatases BVP and PIR1. Structural elements of the Mcel RNA triphosphatase are shown at the top with α helices depicted as boxes and β strands as arrows. The P loop containing the active site cysteine nucleophile is highlighted in the shaded box. Amino acids within the P loop that are essential for the RNA triphosphatase activity of Mcel are denoted by dots above the sequence.

FIG. 3 shows structural conservation among fungal RNA triphosphatases. The amino acid sequences of the catalytic domains of fungal RNA triphosphatases S. cerevisiae Cet1 , C. albicans CaCet1 , S. cerevisiae Cth1, and S. pombe Pct1 are aligned. Gaps in the alignment are indicated by dashes. The β strands that form the triphosphate tunnel of Cet1 are denoted above the sequence. Hydrophilic amino acids that comprise the active site within the tunnel are denoted by dots. Conserved motifs A (β1), B (β9) and C (β11) that define the metal-dependent RNA triphosphatase family are indicated below the sequence. The amino acid sequence of P. falciparum Pgt1 is aligned to those of the four fungal triphosphatases. Peptide segments with the highest degree of conservation in all five proteins are highlighted by the shaded boxes. The poly-asparagine insert in Pgt1 is omitted from the alignment and is denoted by a triangle under the sequence between strands β6 and β7.

FIG. 4 shows the amino acid sequence of the Plasmodium falciparum RNA guanylyltransferase Pgt1. The six nucleotidyl transferase motifs in the guanylyltransferase are highlighted in shaded boxes.

FIG. 5 shows the purification and guanylyltransferase activity of Plasmodium falciparum Pgt1. FIG. 5A shows Pgt1 purification. Aliquots (15 μl) of the soluble bacterial lysate (L) Ni-agarose flow-through (FT), wash (W) and the indicated imidazole eluates were analyzed by SDS-PAGE. The fixed gel was stained with Coomassie blue dye. The positions and sizes (in kDa) of marker polypeptides are shown on the left. FIG. 5B shows guanylyltransferase activity of Pgt1. Reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 5 mM MgCl 2 , 0.17 μM [α 32 P]GTP, and 1 μl of the protein fractions specified above the lanes were incubated at 30° C. for 10 min. The reaction products were resolved by SDS-PAGE. An autoradiograph of the dried gel is shown. The positions and sizes (in kDa) of marker polypeptides are indicated on the left.

FIG. 6 show the dependence of guanylyltransferase activity on enzyme concentration and divalent cation concentration. FIG. 6A shows protein titration. Reaction mixtures (20 μl) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 5 mM MgCl 2 , 0.17 μM [α 32 P]GTP, and Pgt1 as specified were incubated at 30° C. for 10 min. The reaction was quenched with SDS and the products were resolved by SDS-PAGE. The extent of Pgt1-[ 32 P]GMP formation was quantitated by scanning the gel with a Fujix Phosphorimager and is plotted as a function of input protein. FIG. 6B shows divalent cation requirement. Reaction mixtures (20 μl) containing 50 mM Tris HCl (pH 8.0), 5 mM DTT, 0.17 μM [α 32 P]GTP, 200 ng of Pgt1, and either MgCl 2 or MnCl 2 as specified were incubated at 30° C. for 10 min. The extent of Pgt1-[ 32 P]GMP formation is plotted as a function of divalent cation concentration.

FIG. 7 shows a kinetic analysis of Pgt1-GMP formation. Reaction mixtures (100 μl) containing 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MnCl 2 , 1 μg of Pgt1, and either 2.5, 5, or 10 μM [α 32 P]GTP were incubated at 30° C. The reaction was initiated by adding Pgt1. Aliquots (10 μl) were withdrawn at the times indicated and quenched immediately with SDS. Pgt1-[ 32 P]GMP formation is plotted as a function of time.

FIG. 8 shows glycerol gradient sedimentation of P. falciparum guanylyltransferase. An aliquot of the Ni-agarose fraction of Pgt1 (50 μg of protein) was mixed with marker proteins catalase (50 μg), BSA (50 μg), and cytochrome c (50 μg) and the mixture was applied to a 4.8-ml 15-30% glycerol gradient containing 0.5 M NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM 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. The peaks of the internal marker proteins are indicated by arrowheads. Aliquots (2 μl) of each fraction were assayed for enzyme-GMP formation in a reaction mixture (20 μl) containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 5 mM MnCl 2 , and 5 μM [α 32 P]GTP.

FIG. 9 shows the RNA capping activity of recombinant Pgt1. The isolated Pgt1-[ 32 P]GMP complex was reacted with 5′ diphosphate-terminated poly(A) and the RNA reaction product was deproteinized and then recovered by ethanol-precipitation. Aliquots of the product were treated with nuclease P1 followed by treatment with alkaline phosphatase. The treated samples and an undigested control sample of the reaction product were analyzed by thin-layer chromatography on polyethyleneimine-cellulose plates developed with 0.45 M ammonium sulfate. An autoradiograph of the chromatogram is shown. The chromatographic origin and the positions of GpppA, GTP, and P i are indicated on the right.

FIG. 10 shows the amino acid sequence of Plasmodium falciparum RNA triphosphatase Prt1. The phosphohydrolase motifs A, B and C are highlighted in shaded boxes. The C-terminus of the truncated Prt1-CΔ140 polypeptide is indicated by the dot above the sequence.

FIG. 11 shows the purification and metal-dependent phosphohydrolase activity of P. falciparum RNA triphosphatase. FIG. 11A shows Pgt1 purification. Aliquots (15 μl) 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. FIG. 11B shows manganese-dependent NTP hydrolysis. Phosphohydrolase reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM [γ 32 P]ATP, 2 mM MnCl 2 or MgCl 2 , and 0.6 μg of recombinant Pgt1 (0.1 M imidazole eluate) were incubated for 15 min at 30° C. An aliquot (2.5 μl) of the reaction 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. The positions of [γ 32 P]ATP and 32 P i are indicated.

FIG. 12 shows the ATPase and RNA triphosphatase activities of P. falciparum Prt1. FIG. 12A shows dependence of ATPase activity on protein concentration. Reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM [γ 32 P]ATP (10 nmol of ATP), 2 mM MnCl 2 , and recombinant Pgt1 as specified were incubated for 15 min at 30° C. FIG. 12B shows magnesium-dependent RNA 5′ triphosphatase activity. Reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 2 μM 5′ [γ 32 P]-labeled poly(A) (20 pmol of triphosphate RNA ends), either 2 mM MgCl 2 or no added divalent cation, and recombinant Pgt1 as specified were incubated for 15 min at 30° C. The reaction products were analyzed by TLC and the extent of 32 P i formation was quantitated by scanning the chromatograms with a Phosphorimager. ATPase and RNA triphosphatase activities are plotted as a function of input protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the identification of compounds that inhibits the growth of Plasmodium falciparum and other unicellular eukaryotic parasites by virtue of the effects of said compounds on the capping of parasite mRNA.

The present invention provides isolated DNAs encoding a RNA guanylyltransferase and a RNA 5′ triphosphatase from Plasmodium falciparum , vectors for expression of recombinant RNA guanylyltransferase and RNA 5′ triphosphatase, and purified RNA guanylyltransferase and RNA 5′ triphosphatase having amino acid sequences of SEQ ID No. 1 and 2 respectively.

It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA that encodes the protein. Because of the degeneracy of the genetic code (i.e., for most amino acids, more than one nucleotide triplet (codon) codes for a single amino acid), different nucleotide sequences can code for a particular amino acid, or polypeptide. Thus, the polynucleotide sequences of the subject invention also encompass those degenerate sequences that encode the polypeptides of the subject invention, or a fragment or variant thereof. Accordingly, any nucleotide sequence (mutated from the sequences disclosed herein) which encodes the mRNA capping enzymes described herein comes within the scope of this invention and the claims appended hereto.

Also, as described herein, fragments or mutated versions of the mRNA capping enzymes are an aspect of the subject invention so long as such fragments or mutated versions retain the biochemical activity so that such fragments or mutated versions are useful in the methods described herein. As used herein, “fragment,” as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. As used herein, “mutated version,” as applied to a polypeptide, will ordinarily be an altered form of the polypeptide in which one or more amino acids are substituted by different amino acids or by modified amino acids. Mutated versions can be generated by methods known to those skilled in the art, e.g., by chemical modification of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. The ability of a candidate fragment or mutated version to exhibit a characteristic of the mRNA capping enzymes can be readily assessed by a person having ordinary skill in this art by using the methods described herein.

In one embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of Plasmodium RNA guanylyltransferase, comprising the steps of: a) contacting said Plasmodium RNA guanylyltransferase with guanosine triphosphate and a divalent cation cofactor in the presence or absence of a test compound; and detecting formation of a covalent enzyme-GMP intermediate. A lack of formation of an enzyme-GMP intermediate or a reduction in the formation of said intermediate indicates inhibition of said Plasmodium RNA guanylyltransferase by said test compound. Preferably, the divalent cation cofactor is manganese or magnesium. Detection of an enzyme-GMP intermediate may be by any method readily known to those having ordinary skill in this art; preferable methods include radioisotope assay and fluorescence assay. A representative Plasmodium RNA guanylyltransferase is the RNA guanylyltransferase from Plasmodium falciparum disclosed herein.

In another embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of Plasmodium RNA guanylyltransferase, comprising the steps of: a) contacting said Plasmodium RNA guanylyltransferase with guanosine triphosphate and a divalent cation cofactor and a diphosphate-terminated RNA in the presence or absence of a test compound; and detecting formation of a GMP-capped RNA. A lack of formation of a GMP-capped RNA or a reduction in the formation of said GMP-capped RNA indicates inhibition of said Plasmodium RNA guanylyltransferase by said test compound. Preferably, the divalent cation cofactor is manganese or magnesium. Although detection of a GMP-capped RNA may be by any method readily known to those having ordinary skill in this art, preferable methods include radioisotope assay and fluorescence assay. A representative Plasmodium RNA guanylyltransferase is the RNA guanylyltransferase from Plasmodium falciparum disclosed herein, i.e., Plasmodium guanylyltransferase has the amino acid sequence of SEQ ID No. 1, is a fragment of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1, or is a mutated version of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1.

In yet another embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of unicellular eukaryotic parasite RNA 5′ triphosphatase, comprising the steps of: a) contacting said parasite RNA 5′ triphosphatase with a 5′ triphosphate-terminated RNA or a nucleoside triphosphate and a divalent cation cofactor in the presence or absence of a test compound; and detecting hydrolysis of said 5′ triphosphate-terminated RNA or nucleoside triphosphate. A lack of hydrolysis of said 5′ triphosphate-terminated RNA or nucleoside triphosphate or a reduction in the hydrolysis of said 5′ triphosphate-terminated RNA or nucleoside triphosphate indicates inhibition of said parasite RNA 5′ triphosphatase by said test compound. Preferably, the divalent cation cofactor is magnesium, manganese or cobalt. Although detection of hydrolysis may be by any method readily known to those having ordinary skill in this art, preferable methods include radioisotope assay, calorimetric assay, spectrophotometric assay, and fluorescence assay. A representative parasite RNA triphosphatase is the RNA triphosphatase from Plasmodium falciparum disclosed herein.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. A coding sequence is “operably linked” and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A “cis-element” is a nucleotide sequence, also termed a “consensus sequence” or “motif”, that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

A cell has been “transformed” or “transfected” with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed “transgenic”.

As used herein, the term “host organism” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant, protozoan, and animal cells. A recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis . Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells, insect cells, and plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:

EXAMPLE 1

Identification of Plasmodium falciparum RNA Guanylyltransferase, Pgt1

A candidate Plasmodium falciparum mRNA guanylyltransferase was identified on phylogenetic grounds. The candidate enzyme is a 520 amino acid polypeptide encoded by a continuous ORF on chromosome 14 (FIG. 4 ). 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. The Plasmodium guanylyltransferase (henceforth named Pgt1) contains all six catalytic motifs in the standard order and spacing (FIG. 2 and FIG. 4 ), 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 counterpart in other capping enzymes. The hydrophilic segment is predicted, based on the crystal structure of Chlorella virus guanylyltransferase [9], to comprise a large surface loop. All of the amino acids within the six motifs that are essential for the function of S. cerevisiae RNA guanylyltransferase Cegt1 [10] are conserved in the Plasmodium protein, as are the residue that make direct contact with the GTP substrate in the Chlorella virus guanylyltransferase-GTP cocrystal (FIG. 1 ).

EXAMPLE 2

Pgt1 Expression Vector

A DNA fragment containing the PGT1 open reading frame was amplified by polymerase chain reaction from total P. falciparum genomic DNA using oligonucleotide primers designed to introduce an N deI restriction site at the predicted translation start codon and a XhoI site 3′ of the predicted stop codon. The 1.6-kbp PCR product was digested with N d el and X h oI and inserted into the T7 RNA polymerase-based expression plasmid pET16b to generate plasmid pET-His-Pgt1. The nucleotide sequence of the Plasmodium DNA insert was determined. The predicted amino acid sequence of the 520-amino acid Pgt1 protein encoded by this plasmid is shown in FIG. 4 .

EXAMPLE 3

Production and Purification of Pgt1

Pgt1 protein was produced in bacteria as an N-terminal His 10 -tagged fusion protein. pET-His-Pgt1 was transformed into Escherichia coli BL21-CodonPlus(DE3). A 500-ml culture of E. coli BL21-CodonPlus(DE3)/pET-His-Pgt1 was grown at 37° C. in Luria-Bertani medium containing 0.1 mg/ml ampicillin and 50 μg/ml chloramphenicol until the A 600 reached 0.5. The culture was adjusted to 2% ethanol and then incubated at 17° C. for 24 h. Cells were harvested by centrifugation and the pellet was stored at −80° C. All subsequent procedures were performed at 4° C. Thawed bacteria were resuspended in 50 ml of buffer A (50 mM Tris HCl [pH 7.5], 0.25 M NaCl, 10% sucrose). Cell lysis was achieved by addition of lysozyme and Triton X-100 to final concentrations of 100 μg/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity and insoluble material was removed by centrifugation for 45 min at 17,000 rpm in a Sorvall SS34 rotor.

The His-tag allowed for rapid enrichment of Pgt1 based on the affinity of the tag for immobilized nickel (FIG. 5 A). 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 step-wise 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, and 1 M imidazole. The polypeptide compositions of the column fractions were monitored by SDS-polyacrylamide gel electrophoresis (PAGE). The ˜70 kDa recombinant Pgt1 polypeptide was retained on the column and recovered predominantly in the 0.1 M imidazole fraction (which contained 5 mg of protein). The enzyme preparation was stored at −80° C.

EXAMPLE 4

Demonstration and Characterization of the Guanylyltransferase Activity of Pgt1

Guanylyltransferase activity was measured by reaction of the protein with [α 32 P]GTP in the presence of a divalent cation to form the covalent enzyme-GMP intermediate (FIG. 5 B). Enzyme-guanylate formation was linear with respect to Pgt1 concentration ( FIG. 6A ) and was strictly dependent on a divalent cation cofactor—either manganese or magnesium (FIG. 6 B). Other divalent cations—calcium, cobalt, copper and zinc—did not support guanylyltransferase activity (data not shown).

Pgt1 formed a covalent intermediate with [α 32 P]GTP but was unable to do so with [α 32 P]ATP (not shown). The rate and extent of formation of the covalent intermediate was proportional to GTP concentration and leveled off at ≧10 μM GTP (FIG. 7 and data not shown). Approximately 20% of the input enzyme molecules were labeled with GMP during the reaction with 10 μM GTP and 5 mM MnCl 2 . The reaction with 10 μM GTP displayed pseudo first-order kinetics with an apparent rate constant of 1.4 min −1 .

EXAMPLE 5

Native Size of Pgt1

The native size of Pgt1 was analyzed by glycerol gradient sedimentation with internal standards (FIG. 8 ). The guanylyltransferase activity sedimented as a single peak at 4.5 S, which suggested that Pgt1 is a monomer in solution. The activity profile coincided with the distribution of the Pgt1 polypeptide (not shown).

EXAMPLE 6

Pgt1 Catalyzes Capping of RNA

That Pgt1 is a bona fide capping enzyme was demonstrated by isolating the Pgt1-[ 32 P]GMP intermediate by gel filtration and demonstrating that it catalyzed transfer of the GMP to diphosphate-terminated poly(A) to form a GpppA cap structure (FIG. 9 ).

To form the Pgt1-[ 32 P]GMP intermediate, a reaction mixture (100 μl) containing 50 mM Tris HCl (pH 8.0), 5 mM DTT, 2.5 mM MgCl 2 , 5 μM [α 32 P]GTP, and 10 μg of Pgt1 was incubated for 30 min at 30° C. The mixture was adjusted to 25 mM EDTA and 10% glycerol. The native Pgt1-[ 32 P]GMP complex was resolved from free [α 32 P]GTP by gel filtration through a 1-ml column of Sephadex G-50 that had been equilibrated with buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM DTT, 10% glycerol, 0.05% Triton X-100. Gel filtration was performed at 4° C. Five-drop fractions (˜180 μl) were collected serially and the 32 P elution profile was determined by Cerenkov counting of each fraction.

An aliquot (25 μl) of the gel-filtered Pgt1-[ 32 P]GMP complex (recovered in the void volume of the G-50 column) was incubated for 30 min at 30° C. in a reaction mixture (100 μl) containing 50 mM Tris HCl (pH 8.0), 2 mM MgCl 2 , 5 mM, DTT, and 75 pmol 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 μl of 10 mM Tris HCl (pH 8.0), 1 mM EDTA. Aliquots (4 μl) were digested with 5 μg 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 thin-layer chromatography on polyethyleneimine-cellulose plates developed with 0.45 M ammonium sulfate. The radiolabeled material was visualized by autoradiography.

The TLC analysis showed that the isolated Pgt1-[ 32 P]GMP intermediate transferred the GMP to diphosphate-terminated poly(A) to form a radiolabeled GpppA cap structure that was liberated from the RNA by digestion with nuclease P1 and was resistant to alkaline phosphatase (FIG. 9 ).

EXAMPLE 7

The Monofunctional Plasmodium mRNA Guanylyltransferase is Structurally Distinct from the Bifunctional Metazoan Capping Enzyme

Motif I of Pgt1 (62-KxDGxR-67) contains the lysine nucleophile to which GMP becomes covalently attached during the guanylyltransferase reaction. The position of Lys62 relative to the N-terminus of Pgt1 is typical of the monofunctional guanylyltransferases of fungi and Chlorella virus (where the motif I lysine is located at positions 70, 67, 67, and 84 in the S. cerevisiae, S. pombe, C. albicans , and Chlorella virus enzymes, respectively). The Plasmodium enzyme conspicuously lacks the ˜200-aa N-terminal RNA triphosphatase domain present in metazoan and higher plant capping enzymes. Metazoan RNA triphosphatases belong to a distinct branch of the cysteine phosphatase enzyme superfamily and they are easily identified by their primary structure. No ORF encoding a homolog of the metazoan RNA triphosphatase was found 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 NCBI. Thus, Plasmodium apparently does not encode a metazoan-type mRNA capping enzyme.

EXAMPLE 8

Plasmodium falciparum Encodes a Fungal-type RNA Triphosphatase, Prt1

The similarities between the Plasmodium and fungal guanylyltransferases, and the apparent absence of a metazoan-type RNA triphosphatase in Plasmodium, suggested that P. falciparum might possess a fungal-type RNA triphosphatase.

The S. cerevisiae RNA triphosphatase Cet1 exemplifies a growing family of metal-dependent phosphohydrolases that includes the RNA triphosphatases encoded by other fungi ( Candida albicans and Schizosaccharomyces pombe ), by algal virus PBCV-1, and by several groups of animal viruses (poxviruses, African swine fever virus, and baculoviruses). The yeast/viral triphosphatase family is defined by two glutamate-rich peptide motifs (motifs A and C) that are essential for catalytic activity and comprise the metal binding site and by a basic peptide motif (motif B) that is implicated in binding the 5′ triphosphate moiety of the substrate (FIG. 3 ). The crystal structure of S. cerevisiae RNA triphosphatase reveals that the active site is located within the hydrophilic core of a topologically closed 8-stranded β barrel—the so-called “triphosphate tunnel”. The β strands comprising the tunnel (β1, β5, β6, β7, β8, β9, β10, and β11) are displayed over the Cet1 amino acid sequence shown in FIG. 3 .

A PSI-BLAST search [18] initially identified a short segment of weak similarity between Cet1 and the hypothetical P. falciparum protein PFC0985c encoded on chromosome 3 (BLAST score 42). The similarity between PFC0985c and the other fungal RNA triphosphatases was statistically significant after the first iteration of the search (BLAST score 122).

A DNA fragment containing the ORF was amplified by polymerase chain reaction from total P. falciparum genomic DNA using oligonucleotide primers designed to introduce an N c oI restriction site at the methionine codon and a BamHI site 3′ of the predicted stop codon. The PCR product was digested with N c ol and BamHI and cloned into plasmid pYX132. The nucleotide sequence of the Plasmodium DNA insert was determined. The predicted amino acid sequence of the 596-amino acid putative Plasmodium RNA triphosphatase (henceforth named Prt1) is shown in FIG. 10 .

The Plasmodium and fungal protein sequences were then aligned manually using the tertiary structure of Cet1 and known structure-activity relationships for fungal RNA triphosphatases as a guide. It was thereby possible to identify in the Plasmodium protein counterparts of all eight β strands of the Cet1 triphosphate tunnel (FIG. 3 ). The Plasmodium protein contains a 162-amino acid segment between strands β6 and β7, consisting mainly of poly-asparagine and acidic residues, that has no counterpart in other RNA triphosphatases (FIG. 10 ). Reference to the Cet1 structure suggests that this segment is a surface loop emanating from the roof of the tunnel. The instructive point is that the twelve catalytically important hydrophilic amino acids within the tunnel that comprise the active site of fungal RNA triphosphatases are conserved in the Plasmodium Pgt1 protein (FIG. 3 ).

EXAMPLE 9

Demonstration and Characterization of the Triphosphatase Activity of Prt1

That Pgt1 is a bona fide member of the fungal-type family of metal-dependent RNA triphosphatases was demonstrated by isolating and characterizing a catalytically active recombinant version of the protein.

A deletion mutant PRT1-CΔ140 lacking the C-terminal 140 amino acids was generated by PCR amplification with a primer designed to introduce a new stop codon and BamHI site immediately downstream. The C-terminus of the Prt1-CΔ140 polypeptide is indicated by the dot above the sequence in FIG. 10 . The PCR product was digested with Nc of 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 so as to fuse the ORF in-frame to N-terminal His/Smt3. The expression vector was transformed into Escherichia coli BL 21-CodonPlus(DE3). A 200-ml culture amplified from a single transformant was grown at 37° C. in Luria-Bertani medium containing 60 μg/ml kanamycin and 100 μg/ml chloramphenicol until the A 600 reached 0.5. The culture was adjusted to 2% ethanol and 0.4 mM IPTG and then incubated at 17° C. for 16 h. Cells were harvested by centrifugation. All subsequent procedures were performed at 4° C. Bacteria were resuspended in 10 ml of buffer A (50 mM Tris HCl [pH 7.5], 0.25 M NaCl, 10% sucrose). Cell lysis was achieved by addition of lysozyme and Triton X-100 to final concentrations of 100 μg/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity and insoluble material was removed by centrifugation for 40 min at 16,000 rpm in a Sorvall SS34 rotor.

The His 6 -tag allowed for enrichment of Pgt1 based on the affinity of the His/Smt3 μleader for immobilized nickel (FIG. 11 A). The soluble extract was applied to a 0.8-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 5 ml of the same buffer and then eluted step-wise with 1.5-ml aliquots of buffer (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, and 0.5 M imidazole. The polypeptide compositions of the column fractions were monitored by SDS-polyacrylamide gel electrophoresis (PAGE). The recombinant Pgt1 polypeptide was recovered predominantly in the 0.1 M imidazole fraction (which contained 0.9 mg of protein). The enzyme preparation was stored at—80° C.

Recombinant Pgt1 displayed the signature biochemical feature of the fungal RNA triphosphatase family—it catalyzed the hydrolysis of the γ phosphate of ATP in the presence of manganese (FIG. 11 B). Activity was dependent on a metal cofactor and, as with the fungal enzymes, magnesium was ineffective in supporting ATP hydrolysis by Pgt1(FIG. 11 B). ATPase activity increased with increasing Pgt1 concentration (FIG. 12 A). Pgt1 also catalyzed the metal-dependent hydrolysis of the γ phosphate of triphosphate-terminated RNA (FIG. 12 B). Thus, Pgt1 displays the requisite properties of a component of the Plasmodium mRNA capping apparatus.

EXAMPLE 10

Methods to Identify Inhibitors of Prt1

Pgt1 is the newest member of the fungal/viral family of metal-dependent RNA triphosphatases defined by motifs A, B, and C. Pgt1 is an extremely 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 capping enzyme in humans and in arthropods and (ii) metazoans encode no identifiable homologs of the fungal or Plasmodium RNA triphosphatases. Thus, a mechanism-based inhibitor of Pgt1 should be highly selective for the malaria parasite and have 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 Pgt1 would be effective at all stages of the parasite's life cycle. Also, the structural similarity between Pgt1 and the fungal RNA triphosphatases raises the exciting possibility of achieving antifungal and antimalarial activity with a single class of mechanism-based inhibitors.

All members of this RNA triphosphatase family display magnesium dependent RNA triphosphatase activity. They also display robust nucleoside triphosphatase (NTPase) activity in the presence of manganese or cobalt as the divalent cation cofactor. Mutational analysis indicates that the NTPase function of the fungal/viral enzymes is performed by the same catalytic moieties on the enzyme that carry out the RNA triphosphatase reaction. Therefore, the measurement of either RNA triphosphatase or NTPase activity in vitro is a suitable means to screen for inhibitors of Plasmodium falciparum Prt1. However, assay of the NTP hydrolysis by Pgt1 offers a much more convenient assay than RNA triphosphatase for conducting large scale testing of Prt1 inhibitors. This is because NTPs are commercially available (including radioactively labeled NTPs), whereas the synthesis of triphosphate-terminated RNA is technically complex.

Detection of NTP hydrolysis can entail the use of radiolabeled NTP and product analysis by thin layer chromatography (FIG. 11 B). However, the assay is easily adapted to a non-radioactive calorimetric method of detection of P i release from NTP [19], a spectrophotometric assay for inorganic phosphate [20], or a fluorescence-based detection method [21, 22]. A calorimetric, spectrophotometric, or fluorescence-based assay of Pgt1 activity is especially conducive to high-throughput screening of candidate inhibitors.

EXAMPLE 11

Heuristic Phylogeny of Eukaryotes Based on their Capping Enzymes

Capping enzymes are a good focal point for considering eukaryotic evolution because the mRNA cap structure is ubiquitous in eukaryotic organisms, but absent from the bacterial and archaeal kingdoms. Thus, any differences in the capping apparatus between taxa would reflect events that post-date the emergence of ancestral nucleated cells. The enzymes that catalyze the basic nucleic acid transactions (DNA replication, DNA repair, RNA synthesis, and RNA processing) are generally well conserved in lower and higher eukaryotes. Yet, in the case of the capping apparatus, there is a complete divergence of the triphosphatase component and of the physical linkage of the triphosphatase and guanylyltransferase in unicellular and multicellular organisms.

This suggests a heuristic scheme of eukaryotic phylogeny based on two features of the mRNA capping apparatus: the structure and mechanism of the triphosphatase component (metal-dependent “fungal” type versus metal-independent cysteine-phosphatase type) and whether the triphosphatase is physically linked in cis to the guanylyltransferase component. By these simple criteria relying on “black-and-white” differences in the same metabolic pathway, one arrives at different relationships among taxa than those suggested by comparisons of sequence variations among proteins that are themselves highly conserved in all eukaryotes [23]. 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 classified as Apicomplexa along with other pathogenic parasites Toxoplasma and Cryptosporidia) fall into a different lineage distinguished 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 plants. Assuming that multicellular organisms evolved from unicellular ancestors, it can be envisioned that a gene rearrangement event early in eukaryotic evolution transferred a cysteine-phosphatase domain into the same transcription unit as the guanylyltransferase, leading to creation of the triphosphatase-guanylyltransferase fusion protein that is seen today in multicellular eukaryotes. The fusion presumably allowed for the loss of a Cet1-like enzyme from the early metazoan/plant genome or else the divergence of such a protein to a point that it is no longer discernable as Cet1-like. The alternative (and perhaps less parsimonious) explanation that adheres to the sequence-based scheme would be that plants and metazoans independently experienced this gene fusion in distant branches of the phylogenetic tree.

It is conceivable that, as more eukaryotic genomes are sequenced, some species will be found to encode a Cet1-like triphosphatase fused to a guanylyltransferase, whereas others may encode a cysteine-phosphatase-type RNA triphosphatase that participates in cap formation but is separate from the guanylyltransferase, and yet others may encode a novel class of RNA triphosphatase. Nonetheless, a survey of current unicellular genome databases suggests that other protozoans (including Dictyostelium and the pathogenic parasite Trypanosoma) do indeed have ORFs encoding polypeptides that resemble fungal RNA triphosphatases. Thus, antimalarial inhibitors of Plasmodium RNA triphosphatase may be effective against a battery of other unicellular parasites that cause human disease.

Therefore, the present invention provides an isolated DNA encoding a mRNA capping enzyme GTP:RNA guanylyltransferase of Plasmodium falciparum , wherein the amino acid sequence of said enzyme is shown in SEQ ID No. 1. The present invention also provides a recombinant expression vector comprising this DNA, or a fragment thereof that encodes active guanylyltransferase, wherein said DNA is operably linked to regulatory elements that control the expression of said DNA in a host cell. The present invention also provides a host cell transformed with this vector and an isolated polypeptide, or a fragment thereof that possesses guanylyltransferase activity, wherein said polypeptide is encoded by this DNA or a mutated version of this DNA.

In addition, the present invention is directed to a method of screening for a compound that inhibits the catalytic activity of Plasmodium guanylyltransferase, comprising the steps of: contacting said guanylyltransferase with a guanosine triphosphate substrate and a divalent cation cofactor in the presence or absence of said compound; and detecting formation of a covalent guanylyltransferase-GMP intermediate, wherein a lack of formation of said intermediate or a decrease in formation of said intermediate indicates said compound inhibits the catalytic activity of said guanylyltransferase. Preferably, the divalent cation cofactor is selected from the group consisting of manganese and magnesium and the guanosine triphosphate substrate is selected from the group consisting of radioisotopically-labeled guanosine triphosphate and fluorescence-labeled guanosine triphosphate analogs. Generally, the detection of covalent intermediate formation is by a method selected from the group consisting of radioisotope assay and fluorescence assay. The detection of covalent intermediate formation may be by a method selected from the group consisting of analyzing the reaction products by polyacrylamide gel electrophoresis and applying the reaction products to a filter or other solid support so as to retain the guanylyltransferase-GMP intermediate on said filter or solid support without retaining the GTP substrate or pyrophosphate product. Preferably, the Plasmodium guanylyltransferase has the amino acid sequence of SEQ ID No. 1, is a fragment of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1, or is a mutated version of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1. The compound is selected from the group consisting of chemicals, drugs and proteins.

In addition, the present invention is directed to a method of screening for a compound that inhibits the catalytic activity of Plasmodium guanylyltransferase, comprising the steps of: contacting said Plasmodium guanylyltransferase with a guanosine triphosphate substrate and a divalent cation cofactor and a diphosphate-terminated RNA in the presence or absence of said compound; and detecting formation of a GMP-capped RNA, wherein a lack of formation of said GMP-capped RNA or a decrease in formation of said GMP-capped RNA indicates said compound inhibits the catalytic activity of said guanylyltransferase. The Plasmodium guanylyltransferase may have the amino acid sequence of SEQ ID No. 1, is a fragment of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1, or is a mutated version of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1.

In addition, the present invention is directed to an isolated DNA encoding a mRNA capping enzyme RNA 5′ triphosphatase of Plasmodium falciparum , wherein the amino acid sequence of said enzyme is shown in SEQ ID No. 2. A recombinant expression vector comprising this DNA of claim 19 , or a fragment thereof that encodes active RNA 5′ triphosphatase, wherein said DNA is operably linked to regulatory elements that control the expression of said DNA in a host cell is also provided as is a host cell transformed with this vector.

In addition, the present invention is directed to an isolated polypeptide, or a fragment thereof that possesses RNA 5′ triphosphatase activity, wherein said polypeptide is encoded by the DNA of the present invention or a mutated version thereof.

In addition, the present invention is directed to a method of screening for a compound that inhibits the catalytic activity of the RNA 5′ triphosphatase component of the mRNA capping apparatus of a unicellular eukaryotic parasite, comprising the steps of: contacting said unicellular parasite-encoded RNA 5′ triphosphatase with a nucleoside triphosphate substrate and a divalent cation cofactor in the presence or absence of said compound; and detecting hydrolysis of said nucleoside triphosphate to a nucleoside diphosphate and inorganic phosphate, wherein a lack of hydrolysis of said nucleoside triphosphate or a decrease in hydrolysis of said nucleoside triphosphate indicates said compound inhibits the catalytic activity of said parasite-encoded triphosphatase. The unicellular eukaryotic parasite may be, e.g., Plasmodia, Trypanosoma, Leishmania, Toxoplasma, Cryptosporidia, Giardia, Entamoeba, Trichomonas, and Microsporidia. Representative Plasmodium parasites include Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale , and Plasmodium malariae . The parasite-encoded RNA 5′ triphosphatase may have the amino acid sequence of SEQ ID No. 2, is a fragment of the triphosphatase with the amino acid sequence of SEQ ID No. 2, or is a mutated version of the triphosphatase with the amino acid sequence of SEQ ID No. 2.

Furthermore, the present invention is directed to a method of screening for a compound that inhibits the catalytic activity of the RNA 5′ triphosphatase component of the mRNA capping apparatus of a unicellular eukaryotic parasite, comprising the steps of: contacting said unicellular parasite-encoded triphosphatase with a 5′ triphosphate RNA substrate and a divalent cation cofactor in the presence or absence of said compound; and detecting hydrolysis of said triphosphate RNA to a diphosphate RNA and inorganic phosphate, wherein a lack of hydrolysis of said triphosphate RNA or a decrease in hydrolysis of said triphosphate RNA indicates said compound inhibits the catalytic activity of said parasite-encoded triphosphatase.

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Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

104 1 520 PRT Plasmodium falciparum PEPTIDE Plasmodium falciparum RNA guanylyltransferase Pgt1 1Met Ile Thr Ser Thr Tyr His Pro Gly Glu Lys Ile Glu Asn Glu 5 10 15Phe Leu Lys Glu Lys Ile Arg Ser Lys Ile Asn Glu Met Leu Lys 20 25 30Trp Lys Arg Arg Gly Phe Pro Gly Cys Asn Pro Val Ser Leu Thr 35 40 45Asn His Asn Ile Lys Asn Leu Phe Thr Lys Glu Tyr Leu Ile Cys 50 55 60Glu Lys Thr Asp Gly Val Arg Tyr Phe Leu Phe Ile Ala Ser Asn 65 70 75Thr Thr Phe Leu Ile Asp Arg Asn Tyr Glu Ile Phe Lys Asn Asp 80 85 90Met His Ile Pro Thr Ile Glu Asp Leu Ser Lys Lys Gln Gln Leu 95 100 105Thr Leu Leu Asp Gly Glu Leu Val Glu Asp Ile Ile Tyr Asn Glu 110 115 120Lys Thr Gly Val Glu Glu Lys Lys Ile Val Tyr Leu Ile Tyr Asp 125 130 135Gly Leu Tyr Ile Gln Arg Lys Asp Ile Thr Asn Leu Ser Tyr Phe 140 145 150Glu Arg Leu Thr Asn Val Tyr Asn Tyr Val Ile Thr Pro Leu Lys 155 160 165Lys Tyr Lys Lys Ser Gln Lys Asn Lys Lys Asn Lys Asn Asn Leu 170 175 180Gln Thr Asn His Glu Asn Glu Ser Leu Tyr Ile Glu Leu Asp Glu 185 190 195Lys Asp Asn Ile Lys Lys Arg Lys Ser Asn Leu Asn Asn Met Leu 200 205 210Thr Glu Glu Glu Asn Val Leu Ile Ser His Lys Lys Asn Asp His 215 220 225Pro His Ile Asn Asn Lys Asn Met Asn Ala Val Asn Val Asn Gly 230 235 240Val Asp Val Asn Gly Val Asn Ile Asn Gln Asp Phe Asn Asn His 245 250 255Asn Glu Asn Asn Asn Leu Leu Met Asn Gln Gly Ile Leu Ile Asp 260 265 270Glu Asn Asn Asn Gly Ile Gln Asn Ile Gly Thr Asn Asp Asn Ile 275 280 285Asn Ser Leu Asn Asn Cys Asn Leu Leu Leu Tyr Lys Arg Glu Glu 290 295 300His Arg Glu Glu Lys Glu Tyr Glu Glu Glu Glu Asp Glu Arg Ser 305 310 315Tyr Ser Ser Asp Asp Thr Ala Ser Thr Ile His Glu Glu Glu Ile 320 325 330Pro Phe Glu Ile Tyr Leu Lys Asp Phe Tyr Pro Ile Glu Lys Ile 335 340 345Cys Glu Leu Ile Lys Ile Met Lys Lys Leu Pro His Tyr Ser Asp 350 355 360Gly Ile Ile Phe Thr Pro Leu His Ser Pro Tyr Ile Thr Gly Asn 365 370 375Phe Tyr Glu Leu Leu Lys Trp Lys Pro Leu Asn Leu Asn Thr Val 380 385 390Asp Phe Gly Ile Glu Thr Ile Tyr Asp Glu Tyr Asn Ile Pro Ser 395 400 405Lys Phe Glu Leu Phe Ile Ser Ile Asn Gly Val Arg Thr Ser Tyr 410 415 420Lys Cys Tyr Leu Ala Glu Tyr Gly Asp Val Tyr Lys Glu Leu Leu 425 430 435Gln Leu Ala Ile Ser Asn Lys Ile Ser His Tyr Ile Ile Glu Cys 440 445 450Tyr Tyr Val Ser Lys Asn Ile Phe Ser Ile Cys Lys Gly Glu Asn 455 460 465Gly Arg Glu Gln Lys Val Glu Gly Gly Trp Ile Ala Gln Lys Ile 470 475 480Arg Phe Asp Lys Asn Ile Pro Asn Asp Ile Ser Thr Leu Asn Lys 485 490 495Val Ile Gln Ser Ile Leu Asp Asn Ile Thr Ile Asp Ser Leu Ile 500 505 510Lys Glu Ile Ser Arg Asn Arg Lys Ala Lys 515 520 2 596 PRT Plasmodium falciparum PEPTIDE Plasmodium falciparum RNA triphosphatase Prt1 2Met Val Arg Glu Ala His Glu Leu Leu Asp Gly Ser Arg Pro Ile 5 10 15Pro Ile Asp Lys Ile Thr Tyr Glu Leu Ser Gln Asn Ile Ile Leu 20 25 30Ala Phe Asp Asn His Glu Asn Ile Asn Asn Lys Asp Ile Gln Ile 35 40 45Glu Ile Glu Gly Arg Val Gly Leu Val Ile Asp Lys Asn Lys Asn 50 55 60Arg Ile Lys Leu Pro Ile Asn Thr Asp Ala Ile Ile Glu Asn Asn 65 70 75Tyr Ser Asp Phe Gln Ala Gly Ile Asp Arg Glu Ser Phe Glu Tyr 80 85 90Leu Leu Asp Tyr Phe His Asn Met Thr Leu Lys Lys Arg Leu Ser 95 100 105Ile Arg Asn Asn Asp Asn Asn Asn Asn Asn Met Asp Asn Asn Asn 110 115 120Asn Asn Met Asp Asn Asn Asn Asn Asn Asn Asn Asn Asn Ile His 125 130 135Ile His Asn Ser Gly Asn Asn Thr Asn Gln Thr His Ser Tyr Asp 140 145 150Lys Asn Ala Asp Asp Asn Lys Pro Thr Cys Asn Tyr Ser Tyr Asp 155 160 165Lys Lys Asn Ala Cys Ile Tyr Asp Phe Leu Glu Leu Lys Thr Thr 170 175 180Lys Ser Ile Asp Lys Tyr Tyr Val Ile Lys Asn Asn Asn Ser Arg 185 190 195Ile Arg Thr Thr Thr Tyr Leu Asn Asp Asp Asn Lys Gln Glu Thr 200 205 210Glu Ser Met Met Ile Gln Ser Leu Gln Lys Asp Asn Leu Asn Ile 215 220 225Trp Asn Val Tyr Thr Gly Asn Asn Tyr Asp Tyr Phe Asp Asp Asp 230 235 240Glu Glu Asp Asp Asp Asp Asp Tyr Asn Asn Asn Asn Asn Asn Asn 245 250 255Asn Gly Asp Thr Gly Thr Lys Thr Asn Ile Ala Thr Asn Asn Thr 260 265 270His Gly Leu Thr Thr Ser Lys Ser Gln His Ile Tyr Asn Asn Leu 275 280 285Val Asp Lys Asn Asp Ser Ile Asp Tyr Arg Ile Ser Ile Asn Ile 290 295 300Glu Tyr Thr Lys Pro Ile Ser Lys Leu Tyr Leu Ser Lys Asn Thr 305 310 315Pro Val His Glu Arg Leu Lys Glu Arg Thr Thr Phe Ile Asn Thr 320 325 330Tyr Leu Gly Leu Gln Val Asp Met Thr Lys Ile Lys Thr Lys Asn 335 340 345Asn Glu Leu Tyr Glu Val Glu Ile Glu Ile Pro Ser Lys Thr Ile 350 355 360Phe Lys Ala Met Ser Asn Leu Arg Asn Lys Lys Asp Ser Asn Tyr 365 370 375Leu His Phe Ile Cys Ser Asn Leu Val Asn Asn Ile Arg Gly Ile 380 385 390Cys Ser Gln Leu Asn Val Phe Lys Lys Ser Lys His Met Leu Lys 395 400 405Asn Thr Met Ile Thr Lys Leu Asn Asn Asn Ser Asn Asn Gln Asn 410 415 420Asn Leu Ser Leu Leu Pro Asn His Pro Asn Asp Asp Thr Ile Ser 425 430 435Ser Lys Glu Lys Glu Lys Phe Lys Lys Tyr Ile His Ser Val Leu 440 445 450Pro Ile Val Gly Asp Tyr Met Tyr Arg Val Val Thr Lys Asn Glu 455 460 465Lys His Ile Lys Arg Lys Ile Lys Asp Gln Leu Ile Thr Asn Lys 470 475 480Glu Lys Ile Asn Ile Phe Lys Asn Asn Val Asp Ile Arg Arg His 485 490 495Asn Lys Lys Ser Leu Gln Thr Ile Asn Glu Val His Val Glu Asn 500 505 510Lys Trp Lys Ala Phe Lys Arg Gly Thr Lys Ile Glu Val Leu Leu 515 520 525Cys Ser Asp Asp Glu Glu Tyr Glu Gln Asn Glu Asp Val Gln Asp 530 535 540Ile Asn Asn Glu Tyr Tyr Asp Gln Tyr Lys Asn Glu Glu Asp Thr 545 550 555Ser Leu Tyr Ile Asn Asn Ile Tyr Met His Asn Gln Ile Asn Asn 560 565 570Asn Asn Asn Asn Asn Asn Asp Asn Asp Asn Lys Asn Glu Glu Asn 575 580 585Leu Lys Asn Tyr Lys Asp Phe Tyr Asp Asp Thr 590 595 3 6 PRT Saccharomyces cerevisiae DOMAIN Motif I of RNA guanylyltransferase from Saccharomyces cerevisiae 3Lys Thr Asp Gly Leu Arg 5 4 8 PRT Saccharomyces cerevisiae DOMAIN Motif III of RNA guanylyltransferase from Saccharomyces cerevisiae 4Thr Leu Leu Asp Gly Glu Leu Val 5 5 12 PRT Saccharomyces cerevisiae DOMAIN Motif IIIa of RNA guanylyltransferase from Saccharomyces cerevisiae 5Arg Tyr Leu Met Phe Asp Cys Leu Ala Ile Asn Gly 5 10 6 5 PRT Saccharomyces cerevisiae DOMAIN Motif IV of RNA guanylyltransferase from Saccharomyces cerevisiae 6Asp Gly Leu Ile Phe 5 7 13 PRT Saccharomyces cerevisiae DOMAIN Motif V of RNA guanylyltransferase from Saccharomyces cerevisiae 7Leu Leu Lys Trp Lys Pro Glu Gln Glu Asn Thr Val Asp 5 10 8 10 PRT Saccharomyces cerevisiae DOMAIN Motif VI of RNA guanylyltransferase from Saccharomyces cerevisiae 8Trp Glu Met Leu Arg Phe Arg Asp Asp Lys 5 10 9 6 PRT Schizosaccharomyces pombe DOMAIN Motif I of RNA guanylyltransferase from chizosaccharomyces pombe 9Lys Ser Asp Gly Ile Arg 5 10 8 PRT Schizosaccharomyces pombe DOMAIN Motif III of RNA guanylyltransferase from chizosaccharomyces pombe 10Thr Leu Leu Asp Gly Glu Leu Val 5 11 12 PRT Schizosaccharomyces pombe DOMAIN Motif IIIa of RNA guanylyltransferase from chizosaccharomyces pombe 11Arg Tyr Leu Val Phe Asp Cys Leu Ala Cys Asp Gly 5 10 12 5 PRT Schizosaccharomyces pombe DOMAIN Motif IV of RNA guanylyltransferase from chizosaccharomyces pombe 12Asp Gly Leu Ile Phe 5 13 13 PRT Schizosaccharomyces pombe DOMAIN Motif V of RNA guanylyltransferase from chizosaccharomyces pombe 13Leu Leu Lys Trp Lys Pro Lys Glu Met Asn Thr Ile Asp 5 10 14 10 PRT Schizosaccharomyces pombe DOMAIN Motif VI of RNA guanylyltransferase from chizosaccharomyces pombe 14Trp Arg Phe Leu Arg Phe Arg Asp Asp Lys 5 10 15 6 PRT Candida albicans DOMAIN Motif I of RNA guanylyltra sferase from C. albicans 15Lys Thr Asp Gly Leu Arg 5 16 8 PRT Candida albicans DOMAIN Motif III of RNA guanylyltra sferase from C. albicans 16Thr Leu Leu Asp Gly Glu Leu Val 5 17 12 PRT Candida albicans DOMAIN Motif IIIa of RNA guanylyltra sferase from C. albicans 17Arg Tyr Val Ile Phe Asp Ala Leu Ala Ile His Gly 5 10 18 5 PRT Candida albicans DOMAIN Motif IV of RNA guanylyltra sferase from C. albicans 18Asp Gly Leu Ile Tyr 5 19 13 PRT Candida albicans DOMAIN Motif V of RNA guanylyltra sferase from C. albicans 19Leu Leu Lys Trp Lys Pro Ala Glu Glu Asn Thr Val Asp 5 10 20 10 PRT Candida albicans DOMAIN Motif VI of RNA guanylyltra sferase from C. albicans 20Trp Glu Met Leu Arg Phe Arg Asn Asp Lys 5 10 21 6 PRT Chlorella DOMAIN Motif I of RNA guanylyltransferase fr m Chlorella virus PBCV-1 21Lys Thr Asp Gly Ile Arg 5 22 8 PRT Chlorella DOMAIN Motif III of RNA guanylyltransferase fr m Chlorella virus PBCV-1 22Ser Ile Phe Asp Gly Glu Leu Cys 5 23 12 PRT Chlorella DOMAIN Motif IIIa of RNA guanylyltransferase fr m Chlorella virus PBCV-1 23Ala Phe Val Leu Phe Asp Ala Val Val Val Ser Gly 5 10 24 5 PRT Chlorella DOMAIN Motif IV of RNA guanylyltransferase from Chlorella virus PBCV-1 24Asp Gly Leu Ile Ile 5 25 13 PRT Chlorella DOMAIN Motif V of RNA guanylyltransferase from Chlorella virus PBCV-1 25Leu Phe Lys Leu Lys Pro Gly Thr His His Thr Ile Asp 5 10 26 10 PRT Chlorella DOMAIN Motif VI of RNA guanylyltransferase from Chlorella virus PBCV-1 26Trp Lys Tyr Ile Gln Gly Arg Ser Asp Lys 5 10 27 6 PRT Caenorhabditis elegans DOMAIN Motif I of RNA guanylyltransferase from Caenorhabditis elegans 27Lys Ala Asp Gly Met Arg 5 28 8 PRT Caenorhabditis elegans DOMAIN Motif III of RNA guanylyltransferase from Caenorhabditis elegans 28Thr Leu Val Asp Thr Glu Val Ile 5 29 12 PRT Caenorhabditis elegans DOMAIN Motif IIIa of RNA guanylyltransferase from Caenorhabditis elegans 29Arg Met Leu Ile Tyr Asp Ile Met Arg Phe Asn Ser 5 10 30 5 PRT Caenorhabditis elegans DOMAIN Motif IV of RNA guanylyltransferase from Caenorhabditis elegans 30Asp Gly Leu Ile Phe 5 31 13 PRT Caenorhabditis elegans DOMAIN Motif V of RNA guanylyltransferase from Caenorhabditis elegans 31Val Leu Lys Trp Lys Pro Pro Ser His Asn Ser Val Asp 5 10 32 10 PRT Caenorhabditis elegans DOMAIN Motif VI of RNA guanylyltransferase from Caenorhabditis elegans 32Trp Lys Phe Met Arg Glu Arg Thr Asp Lys 5 10 33 6 PRT Unknown DOMAIN Motif I of RNA guanylyltransferase from mouse 33Lys Ala Asp Gly Thr Arg 5 34 8 PRT Unknown DOMAIN Motif III of RNA guanylyltransferase from mouse 34Thr Leu Leu Asp Gly Glu Met Ile 5 35 12 PRT Unknown DOMAIN Motif IIIa of RNA guanylyltransferase from mouse 35Arg Tyr Leu Ile Tyr Asp Ile Ile Lys Phe Asn Ala 5 10 36 5 PRT Unknown DOMAIN Motif IV of RNA guanylyltransferase from mouse 36Asp Gly Leu Ile Phe 5 37 13 PRT Unknown DOMAIN Motif V of RNA guanylyltransferase from mouse 37Ile Leu Lys Trp Lys Pro Pro Ser Leu Asn Ser Val Asp 5 10 38 10 PRT Unknown DOMAIN Motif VI of RNA guanylyltransferase from mouse 38Trp Val Phe Met Arg Gln Arg Ile Asp Lys 5 10 39 6 PRT Drosophila melanogaster DOMAIN Motif I of RNA guanylyltransferase from Drosophila melanogaster 39Lys Ala Asp Gly Thr Arg 5 40 8 PRT Drosophila melanogaster DOMAIN Motif III of RNA guanylyltransferase from Drosophila melanogaster 40Thr Leu Val Asp Gly Glu Met Val 5 41 12 PRT Drosophila melanogaster DOMAIN Motif IIIa of RNA guanylyltransferase from Drosophila melanogaster 41Arg Tyr Leu Ile Tyr Asp Ile Val Arg Leu Ser Asn 5 10 42 5 PRT Drosophila melanogaster DOMAIN Motif IV of RNA guanylyltransferase from Drosophila melanogaster 42Asp Gly Leu Ile Phe 5 43 13 PRT Drosophila melanogaster DOMAIN Motif V of RNA guanylyltransferase from Drosophila melanogaster 43Val Phe Lys Trp Lys Pro His Glu Leu Asn Ser Val Asp 5 10 44 10 PRT Drosophila melanogaster DOMAIN Motif VI of RNA guanylyltransferase from Drosophila melanogaster 44Trp Asp Phe Met Arg Glu Arg Thr Asp Lys 5 10 45 6 PRT Xenopus laevis DOMAIN Motif I of RNA guanylyltransferase from Xenopus laevis 45Lys Ala Asp Gly Thr Arg 5 46 8 PRT Xenopus laevis DOMAIN Motif III of RNA guanylyltransferase from Xenopus laevis 46Thr Leu Leu Asp Gly Glu Met Ile 5 47 12 PRT Xenopus laevis DOMAIN Motif IIIa of RNA guanylyltransferase from Xenopus laevis 47Arg Tyr Leu Ile Tyr Asp Ile Ile Lys Phe Asn Gly 5 10 48 5 PRT Xenopus laevis DOMAIN Motif IV of RNA guanylyltransferase from Xenopus laevis 48Asp Gly Leu Ile Phe 5 49 13 PRT Xenopus laevis DOMAIN Motif V of RNA guanylyltransferase from Xenopus laevis 49Ile Leu Lys Trp Lys Pro Pro Asn Leu Asn Ser Val Asp 5 10 50 10 PRT Xenopus laevis DOMAIN Motif VI of RNA guanylyltransferase from Xenopus laevis 50Trp Val Phe Met Arg Gln Arg Val Asp Lys 5 10 51 6 PRT Arabidopsis thaliana DOMAIN Motif I of RNA guanylyltransferase from Arabidopsis thaliana 51Lys Ala Asp Gly Thr Arg 5 52 8 PRT Arabidopsis thaliana DOMAIN Motif III of RNA guanylyltransferase from Arabidopsis thaliana 52Thr Leu Leu Asp Gly Glu Met Val 5 53 12 PRT Arabidopsis thaliana DOMAIN Motif IIIa of RNA guanylyltransferase from Arabidopsis thaliana 53Arg Tyr Leu Val Tyr Asp Leu Val Ala Ile Asn Gly 5 10 54 5 PRT Arabidopsis thaliana DOMAIN Motif IV of RNA guanylyltransferase from Arabidopsis thaliana 54Asp Gly Leu Ile Phe 5 55 11 PRT Arabidopsis thaliana DOMAIN Motif V of RNA guanylyltransferase from Arabidopsis thaliana 55Leu Leu Lys Trp Lys Phe Val Glu Thr Leu Asp 5 10 56 10 PRT Arabidopsis thaliana DOMAIN Motif VI of RNA guanylyltransferase from Arabidopsis thaliana 56Trp Val Ser Leu Arg Ile Arg Val Asp Lys 5 10 57 6 PRT Arabidopsis thaliana DOMAIN Motif I of RNA guanylyltransferase from Arabidopsis thaliana 57Lys Ala Asp Gly Thr Arg 5 58 8 PRT Arabidopsis thaliana DOMAIN Motif III of RNA guanylyltransferase from Arabidopsis thaliana 58Thr Leu Leu Asp Gly Glu Met Ile 5 59 12 PRT Arabidopsis thaliana DOMAIN Motif IIIa of RNA guanylyltransferase from Arabidopsis thaliana 59Arg Tyr Leu Ile Tyr Asp Met Val Ala Ile Asn Gly 5 10 60 5 PRT Arabidopsis thaliana DOMAIN Motif IV of RNA guanylyltransferase from Arabidopsis thaliana 60Asp Gly Leu Ile Phe 5 61 13 PRT Arabidopsis thaliana DOMAIN Motif V of RNA guanylyltransferase from Arabidopsis thaliana 61Leu Leu Lys Trp Lys Tyr Pro Glu Met Asn Ser Val Asp 5 10 62 10 PRT Arabidopsis thaliana DOMAIN Motif VI of RNA guanylyltransferase from Arabidopsis thaliana 62Trp Val Ser Met Arg Val Arg Val Asp Lys 5 10 63 6 PRT Trypanosoma brucei gambiense DOMAIN Motif I of RNA guanylyltransferase from Trypanosoma brucei gambiense 63Lys Ala Asp Gly Leu Arg 5 64 8 PRT Trypanosoma brucei gambiense DOMAIN Motif III of RNA guanylyltransferase from Trypanosoma brucei gambiense 64Phe Leu Leu Asp Thr Glu Val Val 5 65 12 PRT Trypanosoma brucei gambiense DOMAIN Motif IIIa of RNA guanylyltransferase from Trypanosoma brucei gambiense 65Asp Phe Ile Tyr Phe Trp Gly Leu Asp Gly Arg Arg 5 10 66 5 PRT Trypanosoma brucei gambiense DOMAIN Motif IV of RNA guanylyltransferase from Trypanosoma brucei gambiense 66Asp Gly Leu Ile Phe 5 67 13 PRT Trypanosoma brucei gambiense DOMAIN Motif V of RNA guanylyltransferase from Trypanosoma brucei gambiense 67Leu Ile Lys Trp Lys Pro Val His Leu Cys Thr Val Asp 5 10 68 10 PRT Trypanosoma brucei gambiense DOMAIN Motif VI of RNA guanylyltransferase from Trypanosoma brucei gambiense 68Trp Thr Phe Arg Asn Ala Arg Asn Asp Lys 5 10 69 6 PRT Crithidia fasciculata DOMAIN Motif I of RNA guanylyltransferase from Crithidia fasciculata 69Lys Val Asp Gly Gln Arg 5 70 8 PRT Crithidia fasciculata DOMAIN Motif III of RNA guanylyltransferase from Crithidia fasciculata 70Trp Met Leu Asp Ala Glu Leu Ser 5 71 12 PRT Crithidia fasciculata DOMAIN Motif IIIa of RNA guanylyltransferase from Crithidia fasciculata 71Asp Tyr Val Phe Phe Gly Gly Lys Gln Ala Lys Arg 5 10 72 5 PRT Crithidia fasciculata DOMAIN Motif IV of RNA guanylyltransferase from Crithidia fasciculata 72Asp Gly Leu Val Phe 5 73 13 PRT Crithidia fasciculata DOMAIN Motif V of RNA guanylyltransferase from Crithidia fasciculata 73Leu Leu Lys Trp Lys Pro Leu Ser Leu Cys Thr Ala Asp 5 10 74 10 PRT Crithidia fasciculata DOMAIN Motif VI of RNA guanylyltransferase from Crithidia fasciculata 74Trp Arg Leu His Arg Leu Arg Ser Asp Lys 5 10 75 6 PRT Unknown DOMAIN Motif I of RNA guanylyltransferase from African swine fever virus 75Lys Ala Asp Gly Ile Arg 5 76 8 PRT Unknown DOMAIN Motif III of RNA guanylyltransferase from African swine fever virus 76Thr Ile Leu Asp Gly Glu Phe Met 5 77 12 PRT Unknown DOMAIN Motif IIIa of RNA guanylyltransferase from African swine fever virus 77Glu Phe Tyr Gly Phe Asp Val Ile Met Tyr Glu Gly 5 10 78 5 PRT Unknown DOMAIN Motif IV of RNA guanylyltransferase from African swine fever virus 78Asp Gly Ile Ile Leu 5 79 13 PRT Unknown DOMAIN Motif V of RNA guanylyltransferase from African swine fever virus 79Thr Phe Lys Trp Lys Pro Thr Trp Asp Asn Thr Leu Asp 5 10 80 10 PRT Unknown DOMAIN Motif VI of RNA guanylyltransferase from African swine fever virus 80Trp Glu Ile Val Lys Ile Arg Glu Asp Arg 5 10 81 6 PRT Unknown DOMAIN Motif I of RNA guanylyltransferase from AcNPV baculovirus 81Lys Leu Asp Gly Met Arg 5 82 8 PRT Unknown DOMAIN Motif III of RNA guanylyltransferase from AcNPV baculovirus 82Val Ala Phe Gln Cys Glu Val Met 5 83 12 PRT Unknown DOMAIN Motif IIIa of RNA guanylyltransferase from AcNPV baculovirus 83Asn Arg Thr Gln Tyr Glu Cys Gly Val Asn Ala Ser 5 10 84 5 PRT Unknown DOMAIN Motif IV of RNA guanylyltransferase from AcNPV baculovirus 84Asp Gly Tyr Val Val 5 85 11 PRT Unknown DOMAIN Motif V of RNA guanylyltransferase from AcNPV baculovirus 85Tyr Val Lys Tyr Lys Trp Met Pro Thr Thr Glu 5 10 86 10 PRT Unknown DOMAIN Motif VI of RNA guanylyltransferase from AcNPV baculovirus 86Ile Asn Val Leu Lys His Arg Arg Asp Arg 5 10 87 6 PRT Plasmodium falciparum DOMAIN Motif I of RNA guanylyltransferase from Plasmodium falciparum 87Lys Thr Asp Gly Val Arg 5 88 8 PRT Plasmodium falciparum DOMAIN Motif III of RNA guanylyltransferase from Plasmodium falciparum 88Thr Leu Leu Asp Gly Glu Leu Val 5 89 12 PRT Plasmodium falciparum DOMAIN Motif IIIa of RNA guanylyltransferase from Plasmodium falciparum 89Val Tyr Leu Ile Tyr Asp Gly Leu Tyr Ile Gln Arg 5 10 90 5 PRT Plasmodium falciparum DOMAIN Motif IV of RNA guanylyltransferase from Plasmodium falciparum 90Asp Gly Ile Ile Phe 5 91 13 PRT Plasmodium falciparum DOMAIN Motif V of RNA guanylyltransferase from Plasmodium falciparum 91Leu Leu Lys Trp Lys Pro Leu Asn Leu Asn Thr Val Asp 5 10 92 10 PRT Plasmodium falciparum DOMAIN Motif VI of RNA guanylyltransferase from Plasmodium falciparum 92Trp Ile Ala Gln Lys Ile Arg Phe Asp Lys 5 10 93 195 PRT Unknown DOMAIN RNA triphosphatase domain of mammalian capping enzyme Mce1 93Lys Ile Pro Pro Arg Trp Leu Asn Cys Pro Arg Arg Gly Gln Pro 5 10 15Val Ala Gly Arg Phe Leu Pro Leu Lys Thr Met Leu Gly Pro Arg 20 25 30Tyr Asp Ser Gln Val Ala Glu Glu Asn Arg Phe His Pro Ser Met 35 40 45Leu Ser Asn Tyr Leu Lys Ser Leu Lys Val Lys Met Ser Leu Leu 50 55 60Val Asp Leu Thr Asn Thr Ser Arg Phe Tyr Asp Arg Asn Asp Ile 65 70 75Glu Lys Glu Gly Ile Lys Tyr Ile Lys Leu Gln Cys Lys Gly His 80 85 90Gly Glu Cys Pro Thr Thr Glu Asn Thr Glu Thr Phe Ile Arg Leu 95 100 105Cys Glu Arg Phe Asn Glu Arg Ser Pro Pro Glu Leu Ile Gly Val 110 115 120His Cys Thr His Gly Phe Asn Arg Thr Gly Phe Leu Ile Cys Ala 125 130 135Phe Leu Val Glu Lys Met Asp Trp Ser Ile Glu Ala Ala Val Ala 140 145 150Thr Phe Ala Gln Ala Arg Pro Pro Gly Ile Tyr Lys Gly Asp Tyr 155 160 165Leu Lys Glu Leu Phe Arg Arg Tyr Gly Asp Ile Glu Glu Ala Pro 170 175 180Pro Pro Pro Val Leu Pro Asp Trp Cys Phe Glu Asp Glu Asp Glu 185 190 195 94 197 PRT C elegans DOMAIN RNA triphosphatase domain of C elegans RNA capping enzyme 94Gly Leu Pro Asp Arg Trp Leu His Cys Pro Lys Thr Gly Thr Leu 5 10 15Ile Asn Asn Leu Phe Phe Pro Phe Lys Thr Pro Leu Cys Lys Met 20 25 30Tyr Asp Asn Gln Ile Ala Glu Arg Arg Tyr Gln Phe His Pro Ala 35 40 45Glu Val Phe Ser His Pro His Leu His Gly Lys Lys Ile Gly Leu 50 55 60Trp Ile Asp Leu Thr Asn Thr Asp Arg Tyr Tyr Phe Arg Glu Glu 65 70 75Val Thr Glu His Glu Cys Ile Tyr His Lys Met Lys Met Ala Gly 80 85 90Arg Gly Val Ser Pro Thr Gln Glu Asp Thr Asp Asn Phe Ile Lys 95 100 105Leu Val Gln Glu Phe His Lys Lys Tyr Pro Asp Arg Val Val Gly 110 115 120Val His Cys Thr His Gly Phe Asn Arg Thr Gly Phe Leu Ile Ala 125 130 135Ala Tyr Leu Phe Gln Val Glu Glu Tyr Gly Leu Asp Ala Ala Ile 140 145 150Gly Glu Phe Ala Glu Asn Arg Gln Lys Gly Ile Tyr Lys Gln Asp 155 160 165Tyr Ile Asp Asp Leu Phe Ala Arg Tyr Asp Pro Thr Glu Asp Asp 170 175 180Lys Ile Leu Ala Pro Glu Lys Pro Asp Trp Glu Arg Glu Met Ser 185 190 195Ile Gly 197 95 196 PRT Drosophila melanogaster DOMAIN RNA triphosphatase domain of Drosophila melanogaster RNA capping enzyme 95Pro Leu Pro Asn Arg Trp Leu Tyr Cys Pro Arg Lys Ser Asp Thr 5 10 15Ile Ile Ala Glu Arg Phe Leu Ala Phe Lys Thr Pro Leu Ser Asn 20 25 30Asn Phe His Asp Lys Met Pro Ile Glu Cys Thr Phe Gln Pro Glu 35 40 45Met Leu Phe Glu Tyr Cys Lys Thr Leu Lys Val Lys Leu Gly Leu 50 55 60Trp Val Asp Leu Thr Asn Thr Lys Arg Phe Tyr Asp Arg Ser Ala 65 70 75Val Glu Glu Leu Gly Ala Lys Tyr Ile Lys Leu Gln Cys Arg Gly 80 85 90His Gly Glu Thr Pro Ser Pro Glu Gln Thr His Ser Phe Ile Glu 95 100 105Ile Val Asp Asn Phe Ile Asn Glu Arg Pro Phe Asp Val Ile Ala 110 115 120Val His Cys Thr His Gly Phe Asn Arg Thr Gly Phe Leu Ile Val 125 130 135Cys Tyr Leu Val Glu Arg Leu Asp Cys Ser Val Ser Ala Ala Leu 140 145 150Ala Ile Phe Ala Ser Ala Arg Pro Pro Gly Ile Tyr Lys Gln Asp 155 160 165Tyr Ile Asn Glu Leu Tyr Lys Arg Tyr Glu Asp Thr Asn Ala Ala 170 175 180Pro Ala Ala Pro Glu Gln Pro Asn Trp Cys Leu Asp Tyr Asp Asp 185 190 195Gly196 96 195 PRT Xenopus laevis DOMAIN RNA triphosphatase domain of Xenopus laevis RNA capping enzyme 96Lys Ile Pro Pro Arg Trp Leu Asn Cys Pro Arg Arg Gly Gln Pro 5 10 15Val Ala Gly Arg Phe Leu Pro Leu Lys Thr Ile Leu Gly Pro Arg 20 25 30Tyr Asp Ser Gln Val Ala Glu Glu Asn Arg Phe His Pro Ser Met 35 40 45Leu Ser Asn Tyr Leu Lys Ser Leu Lys Val Lys Met Gly Leu Leu 50 55 60Val Asp Leu Thr Asn Thr Ser Arg Phe Tyr Asp Arg Asn Asp Ile 65 70 75Glu Lys Glu Gly Ile Lys Tyr Ile Lys Leu Gln Cys Lys Gly His 80 85 90Gly Glu Cys Pro Thr Thr Glu Asn Thr Glu Thr Phe Ile Arg Leu 95 100 105Cys Glu Arg Phe Asn Glu Arg Asn Pro Pro Glu Leu Ile Gly Val 110 115 120His Cys Thr His Gly Phe Asn Arg Thr Gly Phe Leu Ile Cys Ala 125 130 135Phe Leu Val Glu Lys Met Asp Trp Ser Ile Glu Ala Ala Val Ala 140 145 150Thr Phe Ala Gln Ala Arg Pro Pro Gly Ile Tyr Lys Gly Asp Tyr 155 160 165Leu Lys Glu Leu Phe Arg Arg Tyr Gly Asp Ile Glu Asp Ala Pro 170 175 180Lys Pro Pro Glu Leu Pro Asp Trp Cys Phe Glu Glu Glu Asp Val 185 190 195 97 197 PRT Arabidopsis thaliana DOMAIN RNA triphosphatase domain of Arabidopsis thaliana RNA capping enzyme 97Lys Ile Pro Gln Gly Trp Leu Asp Cys Pro Pro Ser Gly Asn Glu 5 10 15Ile Gly Phe Leu Val Pro Ser Lys Val Pro Leu Asn Glu Ser Tyr 20 25 30Asn Asn His Val Pro Pro Gly Ser Arg Tyr Ser Phe Lys Gln Val 35 40 45Ile His Asn Gln Arg Ile Ala Gly Arg Lys Leu Gly Leu Val Ile 50 55 60Asp Leu Thr Asn Thr Thr Arg Tyr Tyr Ser Thr Thr Asp Leu Lys 65 70 75Lys Glu Gly Ile Lys His Val Lys Ile Ala Cys Lys Gly Arg Asp 80 85 90Ala Val Pro Asp Asn Val Ser Val Asn Ala Phe Val Asn Glu Val 95 100 105Asn Gln Phe Val Leu Asn Leu Lys His Ser Lys Lys Tyr Ile Leu 110 115 120Val His Cys Thr His Gly His Asn Arg Thr Gly Phe Met Ile Val 125 130 135His Tyr Leu Met Arg Ser Gly Pro Met Asn Val Thr Gln Ala Leu 140 145 150Lys Ile Phe Ser Asp Ala Arg Pro Pro Gly Ile Tyr Lys Pro Asp 155 160 165Tyr Ile Asp Ala Leu Tyr Ser Phe Tyr His Glu Ile Lys Pro Glu 170 175 180Ser Val Ile Cys Pro Ser Thr Pro Glu Trp Lys Arg Ser Thr Glu 185 190 195Leu Asp 197 98 186 PRT Homo sapiens DOMAIN Human RNA-specific 5′ phosphatase PIR1 98His Ile Pro Glu Arg Trp Lys Asp Tyr Leu Pro Val Gly Gln Arg 5 10 15Met Pro Gly Thr Arg Phe Ile Ala Phe Lys Val Pro Leu Gln Lys 20 25 30Ser Phe Glu Lys Lys Leu Ala Pro Glu Glu Cys Phe Ser Pro Leu 35 40 45Asp Leu Phe Asn Lys Ile Arg Glu Gln Asn Glu Glu Leu Gly Leu 50 55 60Ile Ile Asp Leu Thr Tyr Thr Gln Arg Tyr Tyr Lys Pro Glu Asp 65 70 75Leu Pro Glu Thr Val Pro Tyr Leu Lys Ile Phe Thr Val Gly His 80 85 90Gln Val Pro Asp Asp Glu Thr Ile Phe Lys Phe Lys His Ala Val 95 100 105Asn Gly Phe Leu Lys Glu Asn Lys Asp Asn Asp Lys Leu Ile Gly 110 115 120Val His Cys Thr His Gly Leu Asn Arg Thr Gly Tyr Leu Ile Cys 125 130 135Arg Tyr Leu Ile Asp Val Glu Gly Val Arg Pro Asp Asp Ala Ile 140 145 150Glu Leu Phe Asn Arg Cys Arg Gly His Cys Leu Glu Arg Gln Asn 155 160 165Tyr Ile Glu Asp Leu Gln Asn Gly Pro Ile Arg Lys Asn Trp Asn 170 175 180Ser Ser Val Pro Arg Ser 185 99 168 PRT Unknown DOMAIN Baculovirus RNA-specific 5′ phosphatase BVP 99Met Phe Pro Ala Arg Trp His Asn Tyr Leu Gln Cys Gly Gln Val 5 10 15Ile Lys Asp Ser Asn Leu Ile Cys Phe Lys Thr Pro Leu Arg Pro 20 25 30Glu Leu Phe Ala Tyr Val Thr Ser Glu Glu Asp Val Trp Thr Ala 35 40 45Glu Gln Ile Val Lys Gln Asn Pro Ser Ile Gly Ala Ile Ile Asp 50 55 60Leu Thr Asn Thr Ser Lys Tyr Tyr Asp Gly Val His Phe Leu Arg 65 70 75Ala Gly Leu Leu Tyr Lys Lys Ile Gln Val Pro Gly Gln Thr Leu 80 85 90Pro Pro Glu Ser Ile Val Gln Glu Phe Ile Asp Thr Val Lys Glu 95 100 105Phe Thr Glu Lys Cys Pro Gly Met Leu Val Gly Val His Cys Thr 110 115 120His Gly Ile Asn Arg Thr Gly Tyr Met Val Cys Arg Tyr Leu Met 125 130 135His Thr Leu Gly Ile Ala Pro Gln Glu Ala Ile Asp Arg Phe Glu 140 145 150Lys Ala Arg Gly His Lys Ile Glu Arg Gln Asn Tyr Val Gln Asp 155 160 165Leu Leu Ile 168 100 236 PRT Saccharomyces cerevisiae DOMAIN Catalytic domain of RNA triphosphatase from S. cerevisiae Cet1 100Ile Glu Leu Glu Met Lys Phe Gly Val Ile Ile Asp Ala Lys Gly 5 10 15Pro Asp Arg Val Asn Pro Pro Val Ser Ser Gln Cys Val Phe Thr 20 25 30Glu Leu Asp Ala His Leu Thr Pro Asn Ile Asp Ala Ser Leu Phe 35 40 45Lys Glu Leu Ser Lys Tyr Ile Arg Gly Ile Ser Glu Val Thr Glu 50 55 60Asn Thr Gly Lys Phe Ser Ile Ile Glu Ser Gln Thr Arg Asp Ser 65 70 75Val Tyr Arg Val Gly Leu Ser Thr Gln Arg Pro Arg Phe Leu Arg 80 85 90Met Ser Thr Asp Ile Lys Thr Gly Arg Val Gly Gln Phe Ile Glu 95 100 105Lys Arg His Val Ala Gln Leu Leu Leu Tyr Ser Pro Lys Asp Ser 110 115 120Tyr Asp Val Lys Ile Ser Leu Asn Leu Glu Leu Pro Val Pro Asp 125 130 135Asn Asp Pro Pro Glu Lys Tyr Lys Ser Gln Ser Pro Ile Ser Glu 140 145 150Arg Thr Lys Asp Arg Val Ser Tyr Ile His Asn Asp Ser Cys Thr 155 160 165Arg Ile Asp Ile Thr Lys Val Glu Asn His Asn Gln Asn Ser Lys 170 175 180Ser Arg Gln Ser Glu Thr Thr His Glu Val Glu Leu Glu Ile Asn 185 190 195Thr Pro Ala Leu Leu Asn Ala Phe Asp Asn Ile Thr Asn Asp Ser 200 205 210Lys Glu Tyr Ala Ser Leu Ile Arg Thr Phe Leu Asn Asn Gly Thr 215 220 225Ile Ile Arg Arg Lys Leu Ser Ser Leu Ser Tyr 230 235 101 235 PRT Candida albicans DOMAIN Catalytic domain of RNA triphosphatase from Candida albicans CaCet1 101Val Glu Leu Glu Leu Lys Phe Gly Lys Ile Ile Asp Lys Arg Ser 5 10 15Gly Asn Arg Ile Asp Leu Asn Val Val Thr Glu Cys Ile Phe Thr 20 25 30Asp His Ser Ser Val Phe Phe Asp Met Gln Val Glu Glu Val Ala 35 40 45Trp Lys Glu Ile Thr Lys Phe Leu Asp Glu Leu Glu Lys Ser Phe 50 55 60Gln Glu Gly Lys Lys Gly Arg Lys Phe Lys Thr Leu Glu Ser Asp 65 70 75Asn Thr Asp Ser Phe Tyr Gln Leu Gly Arg Lys Gly Glu His Pro 80 85 90Lys Arg Ile Arg Val Thr Lys Asp Asn Leu Leu Ser Pro Pro Arg 95 100 105Leu Val Ala Ile Gln Lys Glu Arg Val Ala Asp Leu Tyr Ile His 110 115 120Asn Pro Gly Ser Leu Phe Asp Leu Arg Leu Ser Met Ser Leu Glu 125 130 135Ile Pro Val Pro Gln Gly Asn Ile Glu Ser Ile Ile Thr Lys Asn 140 145 150Lys Pro Glu Met Val Arg Glu Lys Lys Arg Ile Ser Tyr Thr His 155 160 165Pro Pro Thr Ile Thr Lys Phe Asp Leu Thr Arg Val Ile Gly Asn 170 175 180Lys Thr Glu Asp Lys Tyr Glu Val Glu Leu Glu Ala Gly Val Met 185 190 195Glu Ile Phe Ala Ala Ile Asp Lys Ile Gln Lys Gly Val Asp Asn 200 205 210Leu Arg Leu Glu Glu Leu Ile Glu Val Phe Leu Asn Asn Ala Arg 215 220 225Thr Leu Asn Asn Arg Leu Asn Lys Ile Cys 230 235 102 232 PRT Saccharomyces cerevisiae DOMAIN Catalytic domain of RNA triphosphatase from S. cerevisiae Cth1 102Ile Glu Ile Glu Met Lys Phe Gly Val Ile Thr Asp Lys Arg Thr 5 10 15His Arg Arg Met Thr Pro His Asn Lys Pro Phe Ile Val Gln Asn 20 25 30Arg Asn Gly Arg Leu Val Ser Asn Val Pro Glu Gln Met Phe Ser 35 40 45Ser Phe Gln Glu Leu Leu Arg Ser Lys Ser Glu Asn Pro Ser Lys 50 55 60Cys Ala Pro Arg Val Val Lys Gln Val Gln Lys Tyr Thr Lys Asp 65 70 75Ser Ile Tyr Asn Cys Asn Asn Ala Ser Lys Val Gly Lys Leu Thr 80 85 90Ser Trp Arg Cys Ser Glu Asp Leu Arg Asn Lys Glu Leu Lys Leu 95 100 105Thr Tyr Ile Lys Lys Val Arg Val Lys Asp Phe Leu Ile Arg Tyr 110 115 120Pro Gln Ser Ser Leu Asp Ala Lys Ile Ser Ile Ser Leu Glu Val 125 130 135Pro Glu Tyr Glu Thr Ser Ala Ala Phe Arg Asn Gly Phe Ile Leu 140 145 150Gln Arg Thr Lys Ser Arg Ser Thr Tyr Thr Phe Asn Asp Lys Met 155 160 165Pro Leu His Leu Asp Leu Thr Lys Val Thr Thr Thr Arg Arg Asn 170 175 180Ser His Gln Tyr Thr Ser His Glu Val Glu Val Glu Met Asp Pro 185 190 195Ile Phe Lys Glu Thr Ile Ser Ala Asn Asp Arg Glu Lys Phe Asn 200 205 210Glu Tyr Met Cys Ser Phe Leu Asn Ala Ser Asp Leu Ile Arg Lys 215 220 225Ala Ala Glu Arg Asp Asn Met 230 103 227 PRT Schizosaccharomyces pombe DOMAIN Catalytic domain of RNA triphosphatase from S. pombe Pct1 103Val Glu Ile Glu Ala Lys Leu Gly Thr Leu Ile Asp Leu Glu Thr 5 10 15Gln Asn Arg Phe Glu Phe Pro Val Met Asn Glu Thr Ile Leu Asn 20 25 30Pro Glu Phe Asn Leu Arg Thr Arg Phe Glu Ser Asp Met Thr Ala 35 40 45Ser Glu His Lys Tyr Leu Asn Glu Phe Leu Asn Gln Ala Phe Arg 50 55 60Asp Ser Gln Lys Pro Gly Arg Leu Pro Phe Ala Tyr Lys His Thr 65 70 75Lys Gln Val Asp Leu Phe Tyr Glu Thr Glu Asp Asn Ser Arg Asp 80 85 90Lys Ile Arg Val Ser Lys Asn Gln Ser Asp Asn Gln Val Leu Ala 95 100 105Cys Val Lys Lys Arg Arg Val Ala Asp Leu Phe Leu Tyr Cys Pro 110 115 120Asn Asp Ala Phe Asp Ile Arg Ile Ser Ile Ser Asp Glu Leu Pro 125 130 135Val Ser Met Pro Ser Gly Asn Gln Gln Pro Ser Leu Thr Arg Leu 140 145 150Lys Asp Arg Val Gly Tyr Val His Gln Glu Ile Lys Ile Asp Leu 155 160 165Thr Lys Thr Thr Gln Asn Asp Pro Val Tyr Asp Thr Thr Glu Arg 170 175 180His Glu Leu Glu Val Glu Phe Gly Asn Ile Ala Asp Leu Arg Asp 185 190 195Arg Ala Gln Lys Ala Lys Asp Gly Met Glu Ala Pro Leu Phe Arg 200 205 210Arg Val Gln Leu Phe Met Asp Asn Val Arg Ile Leu Arg Arg Glu 215 220 225His Ser 227 104 193 PRT Plasmodium falciparum DOMAIN Catalytic domain of RNA triphosphatase from Plasmodium falciparum RNA triphosphatase Prt1 104Ile Glu Ile Glu Gly Arg Val Gly Leu Val Ile Asp Lys Asn Lys 5 10 15Asn Arg Ile Lys Leu Pro Ile Asn Thr Asp Ala Ile Ile Glu Asn 20 25 30Asn Tyr Ser Asp Phe Gln Ala Gly Ile Asp Arg Glu Ser Phe Glu 35 40 45Tyr Leu Leu Asp Tyr Phe His Asn Met Thr Leu Lys Lys Arg Leu 50 55 60Ser Ile Arg Asn Asn Asn Asn Thr His Gly Leu Thr Thr Ser Lys 65 70 75Ser Gln His Ile Tyr Asn Asn Leu Val Asp Lys Asn Asp Ser Ile 80 85 90Asp Tyr Arg Ile Ser Ile Asn Ile Glu Tyr Thr Lys Pro Ile Ser 95 100 105Lys Leu Tyr Leu Ser Lys Asn Thr Pro Val His Glu Arg Leu Lys 110 115 120Glu Arg Thr Thr Phe Ile Asn Thr Tyr Leu Gly Leu Gln Val Asp 125 130 135Met Thr Lys Ile Lys Thr Lys Asn Asn Glu Leu Tyr Glu Val Glu 140 145 150Ile Glu Ile Pro Ser Lys Thr Ile Phe Lys Ala Met Ser Asn Leu 155 160 165Arg Asn Lys Lys Asp Ser Asn Tyr Leu His Phe Ile Cys Ser Asn 170 175 180Leu Val Asn Asn Ile Arg Gly Ile Cys Ser Gln Leu Asn 185 190

Claims

1. An isolated DNA encoding a mRNA capping enzyme RNA 5′ triphosphatase of Plasmodium falciparum, wherein the amino acid sequence of said enzyme is shown in SEQ ID No.2.

2. A recombinant expression vector comprising the DNA of claim 1, or a fragment thereof that encodes active RNA 5′ triphosphatase, wherein said DNA is operably linked to regulatory elements that control the expression of said DNA in a host cell.

3. A host cell transformed with the vector of claim 2.

4. A method for producing mRNA capping enzyme RNA 5′ triphosphatase of SEQ ID NO:2 comprising culturing the host cell of claim 3 under conditions in which said enzyme is expressed, and isolating the enzyme.