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Author's personal copy Molecular & Biochemical Parasitology 176 (2011) 58–63
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Molecular & Biochemical Parasitology
Short communication
A novel dual Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity Jatin Mehta, Renu Tuteja ∗ Malaria Group, International Centre for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India
a r t i c l e
i n f o
Article history: Received 11 August 2010 Received in revised form 6 December 2010 Accepted 9 December 2010 Available online 17 December 2010 Keywords: DEAD box Helicase Malaria parasite Plasmodium falciparum RNA binding Unwinding
a b s t r a c t Helicases are ubiquitous essential enzymes which have significant role in the nucleic acid metabolism. Using in silico approaches in the recent past we have identified a number of helicases in the Plasmodium falciparum genome. In the present study we report purification and detailed characterization of a novel helicase from P. falciparum. Our results indicate that this helicase is a homologue of Dbp5 and DDX19 from yeast and human, respectively. The biochemical characterization shows that it contains DNA and RNA unwinding, nucleic acid dependent ATPase and RNA binding activities. It is interesting to note that this enzyme can unwind DNA duplexes in both 5 to 3 and 3 to 5 directions. Using truncated derivatives we further show that Q motif is essentially required for all of its activities. These studies should make an important contribution in understanding the enzymes involved in nucleic acid metabolism in the parasite. © 2010 Elsevier B.V. All rights reserved.
Helicases are motor proteins, which couple chemical energy of the NTP hydrolysis to move along and unwind double-stranded nucleic acids [1]. Helicases are ubiquitously present in prokaryotes, eukaryotes and viruses and this in turn is reflected by the important roles they play in DNA and RNA metabolic processes [1,2]. DNA and RNA helicases both use the energy of NTP hydrolysis for the purpose of unwinding the duplex regions. RNA helicases have been implicated in other functions like annealing RNA strands, RNP remodelling and stabilizing on-pathway folding intermediates [3]. DEAD box helicases belong to the SF2 superfamily and constitute the largest family of RNA helicases [4]. DEAD box helicases are named so due to the presence of conserved amino acid sequence ‘DEAD’ (Asp-Glu-Ala-Asp) in their motif II. These helicases are characterized by the presence of nine conserved motifs arranged in a collinear fashion along the length of the proteins. In the DEAD box helicases, the conserved motifs are clustered in the central core region whereas, the N and C terminal extensions are highly variable in length and sequence composition [4,5]. These highly divergent flanking N and C terminal regions along with the intervening sequences are thought to provide functional specificity through their interaction with substrate and other interacting partners [2,6]. Dbp5/DDX19 is an evolutionary conserved ATP dependent RNA helicase belonging to the DEAD box family of proteins [7]. It is required for the export of mRNA from the nucleus
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and recently it has been identified as a major player in the process of transcription termination [7,8]. Malaria caused by the Anopheles mosquito-transmitted parasite Plasmodium is one of the leading causes of mortality and morbidity in more than 100 tropical and sub tropical countries of the world [9]. Plasmodium falciparum is the most dangerous of the four species of Plasmodium and causes the most lethal form of malaria [9]. It is estimated that 350–500 million clinical malaria cases occur annually, resulting in 1–3 million deaths each year [9,10]. In the absence of clinically proven vaccine against malaria, there are only a limited number of drugs in widespread use for the treatment of malaria [10]. Compounding this paucity of drugs is the rapid development of resistance of the parasite to standard anti-malarial drugs, leading to doubling in the number of deaths from malaria in many parts of sub-saharan Africa [11]. Therefore the rational development of novel pharmacophores for the purpose of malaria intervention requires the identification of new chemotherapeutic targets. The completed genome of P. falciparum along with highthroughput proteomic approaches has opened new avenues for the identification of new drug and vaccine targets [12,13]. Previous studies have shown that the inhibition of the helicases in vivo by dsRNA and DNA intercalating drugs leads to the death of the parasite [14,15]. Therefore helicases can be feasible anti-malarial drug targets. In the present study we report cloning, expression and detailed characterization of a Dbp5/DDX19 homologue designated as PfD66 from P. falciparum. We report that PfD66 is a dual helicase, which can utilize only ATP/dATP as cofactor for its unwinding activity. Besides Mg2+ and Mn2+ , this helicase can also utilize Zn2+ as a
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cofactor. Using truncated derivatives of PfD66 we further report that Q motif is essentially required for all of its enzymatic activity. This study demonstrates the characterization of a novel functionally active Dbp5/DDX19 homologue from P. falciparum. For detailed characterization of this helicase, the PCR amplification was done using genomic DNA as the gene is not interrupted by introns. The P. falciparum helicase gene (from 496 to 2226 bases) was amplified using the forward primer PfD66F1, 5 -CAAGCTTATGCCAAGTGAGGATCTT-3 and the reverse primer PfD66R1, 5 -CCTCGAGTTAATTTTTCAATTTGGTCA-3 . The PCR product was gel purified and cloned into the pGEM-T vector from Promega (Madison, WI, USA) and the clones were sequenced (Macrogen, Korea). The nucleotide sequence was submitted in GenBank and the Accession No. is EF612437. Using the bioinformatics tools we have reported previously that the gene with PlasmoDB number P14 0563 is the bonafide homologue of yeast Dbp5 and mammalian DDX19, respectively [6,16]. The nucleotide sequence encodes a deduced polypeptide with a predicted molecular mass of ∼66 kDa and is thus designated as PfD66. A multiple alignment of complete amino-acid sequence of Dbp5/DDX19 homologues from P. falciparum, Plasmodium vivax, Cryptosporidium parvum, Saccharomyces cerevisiae and Homo sapiens using ClustalW2 (http://www.ebi.ac.uk) [17] revealed that PfD66 aligned contiguously and showed ∼34–58% homology with its counterparts (Fig. S1, Table S1A). The homology increased to ∼38–80% (Table S1B) when only the core region was aligned and dropped to ∼13–28% when only the N-terminal region was aligned (Table S1C). It contains all the conserved helicase motifs but there are few minor variations in the sequences of motif I, Ia and Ib (Fig. S1). In motif I, Thr (T) is replaced by Ser (S), in motif Ia Ala (A) is replaced by S and in motif Ib T is replaced by Lys (K) (Fig. S1). It is interesting to note that the section Asp55 to Ser68 of the N-terminal extension of H. sapiens DDX19 (underlined in Fig. S1) which folded into an ␣helix [18] is absent in the P. falciparum DBP5 homologue (Fig. S1). In recently solved crystal structure of H. sapiens DDX19 it has been reported that this ␣-helix inserts between the conserved domains of the protein and regulates the ATPase activity [18]. Sequences similar to PfD66 were identified in other Plasmodium species and protozoan parasites. Although they showed similar domain organization, differences in the length of the protein was observed between the Plasmodium PfD66 protein and its homologues from other protozoan parasites (Fig. S1 and S2). PfD66 shows homology ranging between 91% and 95% to its other related homologues of Plasmodium species like Plasmodium berghie (PB000472.01.0), P. vivax (PVX 117510), Plasmodium yoelii (PY06529), Plasmodium chaubaudi (PC000054.02.0) and Plasmodium knowlesi (PKH 124710). Dbp5 homologues of both P. falciparum and P. vivax have an unusually large N-terminal as opposed to the homologues of higher eukaryotes (Fig. S2). Moreover, PfD66 homologues are present in other apicomplexan species like Toxoplasma gondii (XP 002369936.1), Theileria parva (XP 765582), Cryptosporidium muris (XP 002142519), Babesia bovis (XP 001609208) and Theleria annulata (XP 954649). The biochemical characterization of PfD66 was important in order to assign it as a bonafide helicase. Therefore for activity analysis of the protein PfD66, the amplified product was cloned into appropriate sites in the bacterial expression vector pET28b. The recombinant his-tagged PfD66 was purified through Ni2+ -NTA affinity chromatography. The SDS-PAGE analysis followed by silver staining of the purified protein showed that it contains no contaminating protein and is a homogeneous preparation (Fig. 1A, lanes 1 and 2). The purified fraction was further checked by western blot analysis using anti-his antibodies and only a single band in the purified fraction was detected (data not shown). This purified preparation was used for all of the assays described in the following sections. The ssDNA-dependent ATPase activity of PfD66
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was checked using standard assay conditions and 30 ng of purified PfD66 enzyme and 1665 Bq [␥-32 P] ATP as a substrate in the presence of 100 ng of M13 ssDNA using the method described previously [19]. The results clearly showed that PfD66 exhibits MgCl2 and ssDNA dependent ATPase activity (Fig. 1B, lane 3) because in the absence of ssDNA or MgCl2 from reaction there was no activity (Fig. 1B, lane 1 and 2). The ATPase reaction using 30 ng of purified PfD66 at different time points was carried out in order to study the time dependence of ATPase activity. The percent release of radioactive phosphate (Pi) from [␥-32 P] ATP showed linearity up to 120 min (Fig. 1C, lane 6). In order to characterize the DNA unwinding activity of PfD66 the standard strand-displacement assay was used [20]. The kinetics of the DNA unwinding reaction of PfD66 using 30 ng of the purified enzyme and optimal assay conditions and ∼1000 cpm of the substrate in buffer having 2 mM ATP, 0.5 mM MgCl2 and 75 mM KCl showed a linear rate up to 60 min (Fig. 1D, lanes 3–6). PfD66 utilised mainly the divalent cation Mg2+ for activity (Fig. 1E, lane 2) but interestingly unwinding activity was also observed in the presence of some of the other cations such as Mn2+ and Zn2+ (Fig. 1E, lanes 3 and 6, respectively). Whereas other divalent cations like Co2+ , Ni2+ , Ca2+ and Ag2+ were unable to support any significant unwinding activity (Fig. 1E, lanes 4, 5, 7 and 8, respectively). DNA is a bipolar molecule with the two strands running in the antiparallel direction. Two different direction-specific DNA duplex substrates were prepared, one specific for the 3 to 5 direction (Fig. 2A) and the other specific for the 5 to 3 direction (Fig. 2B) in order to determine the direction of unwinding by PfD66. The release of the radiolabeled DNA from the substrates shown in Fig. 2A and B indicates the translocation by the enzyme in the 3 to 5 and 5 to 3 directions, respectively. The results of this DNA unwinding assay using these two substrates clearly indicated that the enzyme PfD66 is able to unwind DNA duplex in both the 5 to 3 and 3 to 5 directions. The results further confirmed that although the helicase activity of PfD66 with both of these substrates was concentration dependent (Fig. 2A, lanes 1–4 and Fig. 2B, lanes 1–4) but it was interesting to note that DNA unwinding activity was more with 3 to 5 direction specific substrate (Fig. S3A) as compared to the 5 to 3 direction specific substrate (Fig. S3B). The RNA helicase assay was done by the method described earlier [20] and by using the partially duplex RNA–RNA substrate with two different concentrations of the enzyme (50 ng and 100 ng). The results show clearly that PfD66 has concentration dependent RNAunwinding activity (Fig. 2C, lanes 1 and 2). The ATPase activity of purified PfD66 was tested in the presence of different kinds of nucleic acid to find its preference for the cofactor. The data showed that the PfD66 exhibited less stimulation of ATPase activity in the presence of same amount (100 ng) of M13 ssDNA (lane 8 of Fig. 2D) as compared to P. falciparum total RNA (lane 7 of Fig. 2D). The ATPase activity was stimulated more with RNA as compared to DNA. Out of the four polynucleotides tested poly(A) (lane 6 of Fig. 2D) showed the maximum stimulation of overall ATPase activity in comparison to poly(G), poly(T) or poly(C) (lanes 3, 4 and 5, respectively of Fig. 2D) and Fig. S3C. These results suggest that it is the poly(A) fraction of the RNA which stimulates the enzyme in vivo. In order to check the activity contributed by various domains, two serial truncations (PfDT1 and PfDT2) were made. For PfDT1 the fragment was amplified from the genomic DNA template using the forward primer PfDT1F, 5 -CAAGCTTGCTTATGCATTGCCTATA3 and reverse primer PfD66R1 (described above), whereas for the truncation PfDT2 the fragment was amplified from the same template using the forward primer PfDT2F, 5 CAAGCTTATGTCTTCACAAGTAGAAACT-3 and the same reverse primer PfD66R1. In the first truncation (PfDT1, ∼44 kDa) Q motif was completely deleted and in the second truncation (PfDT2, ∼30 kDa) helicase N-terminal comprising of the motifs Q, I, Ia,
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Fig. 1. (A) A silver-stained gel of purified PfD66. Lane M contains the protein molecular weight marker and lanes 1 and 2 contain 0.5 g of the purified protein from two different batches. (B) ATPase activity of purified PfD66. Lane C, reaction without enzyme; lane 1, reaction without Mg2+ ; lane 2, reaction without single stranded M13mp19 DNA (ssDNA); lane 3, reaction with enzyme in the presence of ssDNA and Mg2+ . (C) Time dependence of ATPase activity of PfD66. The standard reaction contained 100 ng ssDNA, purified protein and 0.5 mM MgCl2 . The time of incubation is mentioned at the top of the autoradiogram and C is the control reaction without enzyme. (D) Time dependence of helicase activity of PfD66. Lane C is reaction without enzyme and lane B is heat denatured substrate. The time of incubation is mentioned at the top of the autoradiogram. The structure of the substrate is shown in the middle. (E) Cation dependence of helicase activity. Lane C is control reaction without enzyme and lane B is heat denatured substrate. Lane 1 is reaction in absence of any cation and lanes 2–8 are reactions in presence of Mg2+ , Mn2+ , Co2+ , Ag2+ , Zn2+ , Ca2+ and Ni2+ , respectively.
Fig. 2. Direction of unwinding of PfD66 (A and B). The structure of the 3 –5 (A) directionality and 5 –3 (B) directionality substrate are mentioned at the top of the panels. In the autoradiogram the enzyme concentration in ng is mentioned at the top of the autoradiogram and lane C is the substrate without enzyme and lane B is heat denatured substrate. (C) RNA helicase activity of PfD66. The structure of the substrate is shown on the left side of the autoradiogram. Asterisk denotes the 32 P-labelled end. C is control without enzyme and B is heat denatured substrate, respectively. Lanes 1 and 2 are two different concentrations of the enzyme. (D) ATPase activity of PfD66. Lane 1 is control reaction without any cofactor and lane 2 is reaction without enzyme. Lanes 3–8 are reactions in presence of poly(G), poly(T), poly(C), poly(A), P. falciparum total RNA and m13MP18 ssDNA, respectively.
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Fig. 3. Characterization of truncated derivatives of PfD66. (A) Schematic drawing showing the various conserved motifs of PfD66, PfDT1 and PfDT2. Open boxes represent the conserved motifs and the name of the motif is written inside the box. (B) The purified proteins expressed in E. coli and visualized by coomassie blue staining. Lane M, the protein molecular weight marker and lane 1, PfDT1 and lane 2, PfDT2. (C) ssDNA dependent ATPase activity of PfD66 and its truncated derivatives. Lane C is control without enzyme and lanes 1–4 are reactions in the presence of PfD66, PfDT1, PfDT2 and BSA, respectively. The position of ATP and Pi are marked on the left hand side of the autoradiogram. (D) DNA helicase activity of PfD66 and its truncated derivatives. Lane C is control without enzyme, lane B is boiled substrate and lanes 1–4 are reactions in the presence of PfD66, PfDT1, PfDT2 and BSA, respectively. (E–G) RNA binding activity of PfD66 and its truncated derivatives. (E) Western blot probed with anti-his antibody. (F) RNA binding activity. Lanes 1–3 in panels E and F are PfD66, PfDT1 and PfDT2, respectively and in panel F, lane B is blank and lane C is BSA. (G) A graphical representation of the data of panel (F).
Ib and II was completely deleted (Fig. 3A). Both of these truncated derivatives were purified and checked for purity using SDS PAGE (Fig. 3B, lanes 1 and 2). Same concentrations (40 ng) of PfD66, PfDT1 and PfDT2 were checked for DNA helicase and ssDNAdependent ATPase activities. The results clearly indicate that only PfD66 showed the ATPase (Fig. 3C, lane 1) and DNA helicase (Fig. 3D, lane 1) activities whereas the truncated proteins, PfDT1 and PfDT2 having the deletion in the N-terminal were ineffective in showing any activity (Fig. 3C, lanes 2 and 3; Fig. 3D, lanes 2 and 3, respectively). In order to check the efficiency of PfD66 and its truncated derivatives to bind to RNA, the RNA binding propensity analysis was performed. The bioinformatics analysis of PfD66 sequence using the program RNA interface residue prediction was done for the protein 3D structure obtained from Swiss-Model program (http://swissmodel.expasy.org). The software available at
http://yayoi.kansai.jaea.go.jp/qbg/kyg/index.php was used to map the residues having high propensity to bind RNA. The amino acids which have the high probability of binding and are present at the interface are labelled in red while the buried amino acids are labelled in deep blue using this interface residue prediction program (Fig. S4A and B). The RNA binding efficiency of PfD66, PfDT1 and PfDT2 was determined using the RNA binding assay as described previously [21]. The experiment was repeated three times and the results were reproducible. The results indicate that PfD66 shows maximum efficiency in binding RNA (Fig. 3F, lane 1) than PfDT1 (Fig. 3F, lane 2) and PfDT2 (Fig. 3F, lane 3). BSA was used as a control, which showed no binding to the RNA (Fig. 3F, lane C). The results were expressed as percentage RNA binding with PfD66 having 100% RNA binding efficiency. The histogram shows that PfDT1 and PfDT2 bind RNA with 58% and 17% RNA binding efficiency as compared to the full length PfD66 (Fig. 3G). An identical
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blot of PfD66, PfDT1 and PfDT2 was probed with anti-his antibody, which confirmed equal loading of protein (Fig. 3E, lanes 1–3). RNA helicases of the DEAD-box family are ubiquitously involved in each step of RNA metabolism. Traditionally RNA helicases (or unwindases) are defined based on their ability to utilize the energy of NTP binding and hydrolysis to unwind RNA duplexes. However, only in a few cases the unwinding activity of these helicases has been demonstrated in vitro [5]. Recently we have reported the genome wide analysis of helicases from P. falciparum and it was reported that the gene with PlasmoDB number PF14 0563 is a homologue of Dbp5/DDX19 [16,22]. Here we report the cloning and characterization of this DEAD-box helicase from P. falciparum. The results confirm that this protein is a homolog of Dbp5/DDX19 and the purified PfD66 is a nucleic acid-dependent ATPase which has DNA helicase, RNA helicase and RNA binding activities. The divalent cation Mg2+ was essential for the unwinding activity of PfD66. Similar findings have been reported in case of other helicases like PfH45 and SV40 large T antigen helicase [20,23]. Pea DNA helicase (PDH120) also prefers Mg2+ as divalent cation for its unwinding activity [24]. It has been shown that divalent metal cation provides a bridge for ATP to fuel unwinding and ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase [25]. Interestingly PfD66 is able to utilise the other divalent cations like Mn2+ and Zn2+ for supporting the unwinding activity. The ATPase activity of Dbp5 is stimulated by the cellular cofactors like Gle1 and IP6 in vivo [26]. Previously we have reported that there are no homologues of Gle1 in P. falciparum [16]. This observation coupled by the experimental result that the ATPase activity of PfD66 is stimulated by poly(A) rich RNA oligonucleotide as compared to DNA and other polynucleotide fractions suggests that PfD66 activity is probably stimulated by the poly(A) rich mRNA fraction of the RNA in vivo. Human DDX19 and yeast Dbp5p are known to bind RNA as shown by the gel retardation studies [7]. PfD66 also exhibits RNA binding activity. The nucleic acid dependent ATPase activity of PfD66 is required for its unwinding activity. Dbp5 is able to unwind short duplex dsRNA substrates in an ATP dependent fashion [26]. Different domains of the helicase protein are known to play different functions like the N-terminal is involved in the ATPase activity and the C-terminal plays a role in the RNA binding. In order to ascertain the role of Q motif and helicase N-terminal in various activities two truncations (PfDT1 and PfDT2) were made. In PfDT1, the Q motif was deleted, whereas in the PfDT2, the N-terminal comprising of few motifs such as Q, I (GSGKT), Ia (PTRELS), Ib (TPGK) and II (DEAD) was completely deleted. The studies with the truncated derivatives of PfD66 showed that the Q Motif and other motifs I, Ia, Ib and II all are important for helicase and ATPase activities and their deletion renders the protein inactive. In PfDT1 deletion of the ‘Q’ motif may directly impair the interaction with ATP and motif ‘I’. In PfDT2 where, Q, I, Ia, Ib and II motifs were completely deleted there is impaired recognition, binding and ATP hydrolysis, thus further abolishing any ATPase or helicase activities. Interestingly it was also observed that all these motifs play a significant role in the process of RNA binding. Although the deletion of the Q motif reduced RNA binding to ∼58%, on further deletion of other motifs the RNA binding was reduced to only ∼17% of the wild type full-length protein. It has been reported previously that Q motif through its interactions with other motifs mainly with motif I is important for ATP-binding and hydrolysis and RNA binding [5]. Motif Ia has also been reported to be involved in nucleic acid binding [27]. Motif II (DEAD) represents a specific form of the ATPase (Walker B) motif and is reported to be responsible for coupling of ATPase and helicase activity [5,27]. In a recent study describing the structure of Dbp5p from S. cerevisae and S. pombe it has been reported that SpDbp5p contains two RecA-like domains that do not interact with each other in the absence of RNA and the N-terminal flanking region of ScDbp5p (M1-E70) is highly unstructured and the region Y71-LR121 including the Q
motif is dynamic [28]. The structure of human homologue DDX19 reveals that its N-terminal region is folded into an ␣-helix that occupies the central cleft between the N-terminal and C-terminal domains and interacts with C-terminal domain [18,28]. Our data are in agreement with the recently solved crystal structure of human DDX19, where it has been shown that both domain 1 and 2 are required for RNA binding [28]. These results collectively suggest that not only Q motif but other motifs I, Ia, Ib and II (GSGKT, PTRELS, TPGK and DEAD) all are essentially required for all the enzymatic activities of this DEAD box protein PfD66. In a recent study involving human DDX19, it has been shown that the presence of the native N terminus is essential for ssRNA concentration-dependent stimulation of ATPase activity [18]. The structure of Dbp5 in complex with RNA and AMPPNP revealed that the N-terminal extension contributes to the ATP-binding site by moving out of the domain cleft and stretching back towards the C-terminal domain [29]. It is well established that the process of bulk export of mRNAs from nucleus to cytoplasm is highly conserved across eukaryotes. The export-competent mRNA ribonucleoprotein (mRNP) complex consists of mRNAs and a number of nucleocytoplasmic shuttling nuclear proteins, including RNA export factors, poly(A)-binding proteins (PABP), DEAD-box protein 5 (Dbp5), and nucleoporins (NUPs) in eukaryotes. Our knowledge of mRNA export mechanisms in malaria parasite is in its infancy. Recently using bioinformatics approaches we have reported the components of mRNA export in P. falciparum [16]. We have also characterized some of these in the recent past [30,31]. In the present study we have reported the detailed biochemical characterization of one of the important component of this complex Dbp5 from P. falciparum. These studies should make an important contribution in understanding the nucleic acid transaction in the malaria parasite P. falciparum. Acknowledgements This work is partially supported by Department of Biotechnology and Defence Research and Development Organization grants. Infra-structural support from the Department of Biotechnology, Government of India is gratefully acknowledged. We thank Arun Pradhan for help with RNA helicase assay. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2010.12.003. References [1] Singleton MR, Dillingham MS, Wigley DB. Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem 2007;76:23–50. [2] Vindigni A. Biochemical, biophysical, and proteomic approaches to study DNA helicases. Mol Biosyst 2007;3:266–74. [3] Bleichert F, Baserga SJ. The long unwinding road of RNA helicases. Mol Cell 2007;27:339–52. [4] Rocak S, Linder P. DEAD-box proteins: the driving forces behind RNA metabolism. Nat Rev Mol Cell Biol 2004;5:232–41. [5] Cordin O, Banroques J, Tanner NK, Linder P. The DEAD-box protein family of RNA helicases. Gene 2006;367:17–37. [6] Tuteja R, Pradhan A. Unraveling the ‘DEAD-box’ helicases of Plasmodium falciparum. Gene 2006;376:1–12. [7] Tseng SS, Weaver PL, Liu Y, Hitomi M, Tartakoff AM, Chang TH. Dbp5p, a cytosolic RNA helicase, is required for poly (A)+ RNA export. EMBO J 1998;17:2651–62. [8] Gross T, Siepmann A, Sturm D, et al. The DEAD-Box RNA helicase Dbp5 functions in translation termination. Science 2007;315:646–9. [9] Tuteja R. Malaria—an overview. FEBS J 2007;274:4670–9. [10] Winstanley PA. Chemotherapy for falciparum malaria: the armoury, the problems and the prospects. Parasitol Today 2000;16:146–53. [11] Sachs J, Malaney P. The economic and social burden of malaria. Nature 2002;415:80–5. [12] Florens L, Washburn MP, Raine JD, et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 2002;419:520–6.
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