Mutational Analyses of Yeast RNA Triphosphatases Highlight a Common Mechanism of Metal-dependent NTP Hydrolysis and a Means of Targeting Enzymes to Pre-mRNAs in Vivo by Fusion to the Guanylyltransferase Component of the Capping Apparatus

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Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metaldependent NTP phosphohydrolases with a unique tunnel active site composed of eight ␤ strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop ( 76 WKGRRARKT 84 ) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75°C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (K i 1.4 M) that binds more avidly to the active site than the ATP substrate (K m 25 M). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.The m 7 GpppN cap structure (cap 0) 1 is a defining feature of eukaryotic mRNA that is required for mRNA stability and efficient translation. The cap is formed by three enzymes. 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 (guanine-N7) methyltransferase (1, 2). The mRNAs of kinetoplastid protozoa such as Trypanosoma and Leishmania contain a unique hypermodified "cap 4" structure, which is derived from the m 7 GpppN cap by co-transcriptional methylation of seven sites within the first four nucleosides of the spliced leader RNA (3-6). Kinetoplastid mRNAs acquire their 5Ј caps via trans-splicing of an RNA leader sequence containing the cap 4 structure. The hypermethylated cap is required for trans-splicing (7-9).RNA guanylyltransferase and RNA triphosphatase components of the kinetoplastid capping apparatus have been identified (10, 11), but the proteins that catalyze the several methylation steps have not. Whereas the Trypanosoma brucei guanylyltransferase is mechanistically and structurally related to the guanylyltransferases of all other eukaryal species (1, 10), the T. brucei RNA triphosphatase (TbCet1) bears no resemblance whatsoever, structurally or mechanistically, to the analogous enzyme of the human host (11, 12). RNA triphosphatase is thereby recommended as a potential drug t...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structure-Function Analysis of Trypanosoma brucei RNA Triphosphatase and Evidence for a Two-metal Mechanism

Gong

Martins

Shuman

2003

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“…Remarkably, the in vivo function of Cet1(276 -549) was restored when it was fused to the guanylyltransferase domain of the mammalian capping enzyme (9). We proposed that the mammalian domain, Mce1(211-597), which binds avidly to the phosphorylated CTD (15,20), can act as a vehicle to deliver the fused RNA triphosphatase to the RNA polymerase II elongation complex (9,21). Also, because Mce1(211-597) is thermostable (unlike Ceg1; see Fig.…”

Section: Cet1mentioning

confidence: 99%

An Essential Function of Saccharomyces cerevisiae RNA Triphosphatase Cet1 Is to Stabilize RNA Guanylyltransferase Ceg1 against Thermal Inactivation

Hausmann

Schwer

et al. 2001

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Saccharomyces cerevisiae RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Here we show that the guanylyltransferase activity of Ceg1 is highly thermolabile in vitro (98% loss of activity after treatment for 10 min at 35°C) and that binding to recombinant Cet1 protein, or a synthetic peptide Cet1(232-265), protects Ceg1 from heat inactivation at physiological temperatures. Candida albicans guanylyltransferase Cgt1 is also thermolabile and is stabilized by binding to Cet1(232-265). In contrast, Schizosaccharomyces pombe and mammalian guanylyltransferases are intrinsically thermostable in vitro and they are unaffected by Cet1(232-265). We show that the requirement for the Ceg1-binding domain of Cet1 for yeast cell growth can be circumvented by overexpression in high gene dosage of a catalytically active mutant lacking the Ceg1-binding site (Cet1(269 -549)) provided that Ceg1 is also overexpressed. However, such cells are unable to grow at 37°C. In contrast, cells overexpressing Cet1(269 -549) in single copy grow at all temperatures if they express either the S. pombe or mammalian guanylyltransferase in lieu of Ceg1. Thus, the cell growth phenotype correlates with the inherent thermal stability of the guanylyltransferase. We propose that an essential function of the Cet1-Ceg1 interaction is to stabilize Ceg1 guanylyltransferase activity rather than to allosterically regulate its activity. We used protein-affinity chromatography to identify the COOHterminal segment of Ceg1 (from amino acids 245-459) as an autonomous Cet1-binding domain.

“…The metazoan RNA triphosphatases do not require a metal cofactor. In contrast, the RNA triphosphatases of fungi and DNA viruses (poxviruses and baculoviruses), which comprise a new family of nucleoside triphosphate phosphohydrolases, are strictly dependent on a divalent cation cofactor (13)(14)(15)(16)(17)(18)(19). There are no mechanistic or structural similarities whatsoever between the metazoan and viral/fungal triphosphatase families (20).…”

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Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase

Martins

Shuman

2000

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Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate. ) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK a of the bridging oxygen of the ␤ phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the ␥ phosphorus. In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK a than does ADP.RNA triphosphatase catalyzes the hydrolysis of the ␥ phosphate of triphosphate-terminated RNA to form a diphosphate end. Two distinct classes of eukaryotic RNA triphosphatases have been described. The RNA triphosphatases of metazoans and plants belong to a superfamily of phosphatases that includes protein-tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases (1-12). The metazoan RNA triphosphatases do not require a metal cofactor. In contrast, the RNA triphosphatases of fungi and DNA viruses (poxviruses and baculoviruses), which comprise a new family of nucleoside triphosphate phosphohydrolases, are strictly dependent on a divalent cation cofactor (13-19). There are no mechanistic or structural similarities whatsoever between the metazoan and viral/fungal triphosphatase families (20). Thus, RNA triphosphatase presents a remarkable case of complete divergence during the transition from fungal to metazoan species.The metazoan RNA triphosphatases are subdivided into two groups: (i) bifunctional enzymes composed of an N-terminal RNA triphosphatase domain and a C-terminal RNA guanylyltransferase domain (1-9) and (ii) monofunctional RNA triphosphatases (10 -12). The bifunctional enzymes are ubiquitous in higher eukaryotes, where they are responsible for forming the 5Ј cap structure of cellular mRNAs. The monofunctional enzymes perform RNA 5Ј modification reactions uncoupled from or unrelated to cap formation, and their true role in RNA metabolism is not understood. The latter category includes BVP, 1 a phosphatase encoded by the Autographa californica baculovirus (10, 11), and the human PIR1 phosphatase (12). Also in the latter category are the alternatively spliced forms of the cellular capping enzymes that retain the RNA triphosphatase domain but lack essential constituents of the guanylyltransferase active site (7-9).The RNA triphosphatase domains of the metazoan and plant capping enzymes contain the signature HCXXGXXR(S/T) phosphate-binding motif of the protein-tyrosine phosphatase/dual specificity protein phosphatase enzyme superfamily (Fig. 1). Protein-tyrosine phosphatases and dual specificity protein phosphatases cat...