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: 9.67). 10/1997; 94(18):9573-8. DOI: 10.1073/pnas.94.18.9573
Source: PubMed


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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Available from: C. Kiong Ho, Jan 08, 2015
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    • "GTP binds between the six-stranded and four-stranded b sheets of the NTase module and induces no large-scale changes in the enzyme, affecting only the conformations of a few side chains in direct contact with the ligand. The defining features of the vaccinia GTase active site are six conserved motifs that interact with the guanine base (motifs IIIa and IV), the ribose hydroxyls (motifs I and III), and the triphosphate moiety (motifs I, V, and VI) of GTP (Shuman and Lima, 2004; Wang et al., 1997). Vaccinia GTase motif I ( 260 KTDGIP 265 ) is located in the loop between b10 and b11 and contains Lys260, the active site lysine nucleophile to which GMP becomes covalently attached (Niles and Christen, 1993). "
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    ABSTRACT: Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m(7)GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.
    Structure 03/2014; 22(3):452-465. DOI:10.1016/j.str.2013.12.014 · 5.62 Impact Factor
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    • "The messenger RNAs (mRNAs) of all metazoan organisms and most eukaryotic viruses possess a 5′ cap structure consisting of a 7-methyl guanosine (m7G) linked via an inverted 5′–5′ triphosphate bridge to the initiating nucleoside of the transcript (1–3), which is itself modified by 2′-O-ribose methylation at the first and often the second transcribed nucleotides. The inverted m7G or cap0 structure marks transcription start sites, and has multiple effects on gene expression, including enhancement of RNA stability, splicing, nucleocytoplasmic transport and translation initiation, as facilitated by interactions with nuclear and cytoplasmic cap binding proteins (4–8). "
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    ABSTRACT: The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.
    Nucleic Acids Research 02/2011; 39(11):4756-68. DOI:10.1093/nar/gkr038 · 9.11 Impact Factor
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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 · 4.94 Impact Factor
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