Abstract
The mRNA capping apparatus of the protozoan parasite Trypanosoma brucei consists of separately encoded RNA triphosphatase and RNA guanylyltransferase enzymes. The triphosphatase TbCet1 is a member of a new family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and the malaria parasite Plasmodium falciparum. The protozoal/fungal enzymes are structurally and mechanistically unrelated to the RNA triphosphatases of metazoans and plants. These results highlight the potential for discovery of broad spectrum antiprotozoal and antifungal drugs that selectively block the capping of pathogen-encoded mRNAs. We propose a scheme of eukaryotic phylogeny based on the structure of RNA triphosphatase and its physical linkage to the guanylyltransferase component of the capping apparatus.
Highlights
Kinetoplastid protozoan parasites of the genus Trypanosoma are major zoonotic pathogens of humans
The cap is formed by three enzymatic reactions: the 5Ј triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; the diphosphate end is capped with GMP by RNA guanylyltransferase; and the GpppN cap is methylated by RNA-methyltransferase [5]
Identification of a Candidate T. brucei RNA Triphosphatase, TbCet1—Metazoan and plant RNA triphosphatases belong to the cysteine phosphate enzyme superfamily, which is defined by the conserved phosphate-binding loop motif HCXXXXXR(S/ T)
Summary
Yeast Expression Vector for T. brucei RNA Triphosphatase—A DNA fragment containing the TbCET1 open reading frame was amplified by polymerase chain reaction from T. brucei genomic DNA (a gift of Vivian Bellofatto, University of Medicine and Dentistry of New Jersey) using oligonucleotide primers designed to introduce an NcoI restriction site at the predicted translation start codon and a BamHI site 3Ј of the predicted stop codon. Expression and Purification of Recombinant TbCet1—The TbCET1 open reading frame was excised from pYX-TbCET1 with NcoI and BamHI, and the 5Ј overhangs were filled in with T4 DNA polymerase. The column was washed with 5 ml of the same buffer and eluted stepwise with 2-ml aliquots of buffer B (50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 10% glycerol, 0.05% Triton X-100) containing 5, 50, 100, 200, and 500 mM imidazole. The 100 mM imidazole eluate fraction (containing 3 mg of protein) was used to characterize the triphosphatase activity of TbCet
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