Article

Phylogeny of mRNA capping enzymes

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/1997; 94(18):9573-8. DOI: 10.1073/pnas.94.18.9573
Source: PubMed

ABSTRACT The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure-function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.

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    • "The original consensus motif III element was defined as uuuDGEuu (where u is an aliphatic side chain). Later studies showed that the aspartate preceding the glycine, although essential when present in ligases or capping enzymes , is not strictly conserved in all nucleotidyltransferases (Wang et al. 1997). Also, the glycine is replaced in some ligases and capping enzymes by alanine, serine, cysteine , or threonine. "
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    ABSTRACT: T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.
    RNA 01/2007; 12(12):2126-34. DOI:10.1261/rna.271706
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    • "This raises two possibilities: either (1) Trl1 has no counterpart of motif III or (2) it has a counterpart of motif III, but its sequence has diverged from the consensus. We noted previously that the aspartate preceding the glycine, though essential when present in ligases or capping enzymes, is not strictly conserved in all nucleotidyltransferases (Wang et al. 1997). Also, the glycine is replaced in some ligases and capping enzymes by alanine, serine, cysteine, or threonine. "
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    ABSTRACT: Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.
    RNA 07/2005; 11(6):966-75. DOI:10.1261/rna.2170305
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    • "Our findings also suggest a similar function for either ␭C-K169/VP1-K176 or ␭C-K188/VP1-K196 of both avian and grass carp reovirus guanylyltransferases. The amino acid context surrounding the putative GMPacceptor site of the mammalian reovirus capping enzyme ( 190 KDLS) is different from the KXDG consensus active-site motif found in many cellular and viral RNA guanylyltransferases (Wang et al., 1997). On the other hand, sequences similar to the ␭2 KDLS sequence, but not to the KXDG consensus sequence, were found to be widely conserved among the RNA guanylyltransferases of other members of the Reoviridae family ( 487 KDLT for bluetongue virus VP4, 542 KDLKS for rotavirus VP3, and 399 KDTS for phytoreovirus gene S5-encoded protein). "
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    ABSTRACT: We have cloned and sequenced the L3 genome segment of avian reovirus strain 1733, which specifies the viral guanylyltransferase protein, lambdaC. The L3 gene is 3907 nucleotides long and encodes, in a single large open-reading frame, a polypeptide of 1285 amino acid residues, with a calculated M(r) of 142.2 kDa. Expression of this gene in a baculovirus/insect cell system produced a recombinant protein that comigrated with reovirion lambdaC and reacted with anti-reovirus polyclonal serum in a Western blot assay. Incubation of recombinant lambdaC with GTP led to the formation GMP-lambdaC complex via a phosphoamide linkage. Interestingly, a 42-kDa amino-terminal proteolytic fragment of recombinant lambdaC protein also exhibited autoguanylylation activity, demonstrating both that this fragment is necessary and sufficient for autoguanylylation activity and that the 100-kDa complementary fragment is expendable for that activity. Comparison of the deduced amino acid sequence of protein lambdaC with those of the mammalian and grass carp reovirus guanylyltransferases revealed that only two of eight lysine residues within the amino-terminal 42-kDa region are conserved. Interestingly, these two lysines match with the lysine residues in the mammalian reovirus capping enzyme proposed to be essential for autoguanylylation activity. Our alignment analysis also showed that the S-adenosyl-l-methionine-binding pocket previously detected in the mammalian reovirus capping enzyme is fully conserved in its avian and grass carp reovirus counterparts, suggesting that all three enzymes have methylase activity.
    Virology 06/2002; 296(2):288-99. DOI:10.1006/viro.2002.1427
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