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Active Site of the Mrna-Capping Enzyme Guanylyltransferase from Saccharomyces Cerevisiae

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Active Site of the Mrna-Capping Enzyme Guanylyltransferase from Saccharomyces Cerevisiae Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6624-6628, July 1994 Biochemistry Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: Similarity to the nucleotidyl attachment motif of DNA and RNA ligases (nucleoidyl trnsfer/covalent catalysis) LUCILLE D. FRESCO* AND STEPHEN BURATOWSKI*t The Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142 Communicated by Phillip A. Sharp, March 8, 1994 ABSTRACT Nasent mRNAchains are capped atthe 5' end The covalent E-GMP intermediate (EnpG) can be conve- by the addiion ofa guanylyl residue to forma G(5')ppp(5')N... niently identified by radioactive labeling of the protein with structure. During the capping reaction, the guanylyltrnerase [a-32P]GTP. (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly Interest in the mechanism ofcapping has focused attention and covalently guanylylated. In this enzyme-GMP (E-GMP) on the E-GMP complex. Cellular mRNA guanylyltrans- termediate, GMP Is linked to the E-amino group of a lysine ferases from human, rat liver, calf thymus, Artemia salina, residue via a phosphoamide bond. Lys-70 was Identid as the wheat germ, and Saccharomyces cerevisiae all form E-GMP GMP attachment ste of the Saccharomyces cerevisiae guanylyl- covalent complexes (reviewed in ref. 3). In addition, cyto- transferase (encoded by the CEGI gene) by guanyylpeptide plasmic viruses encode their own capping enzymes. The sequencing. CEGI genes with substitutions at Lys-70 were guanylyltransferases of vaccinia virus (12, 13), reovirus (11, unable to support viability in yeast and produced proteins that 14, 15), African swine fever virus (16), rotavirus (17), blue- were not guanylylated in vitro. The CEG1 active site exhibits tongue virus (18), and Shope fibroma virus (19) have also sequence similarity to the active sites of viral guanylyltrans- been shown to form E-GMP complexes. The E-GMP com- ferases and polynuceoide ligases, gIng smlity in the plex has been shown to act as a bonafide intermediate in the ofnucleotidyl tner catalyzed by these enzymes. transguanylation reaction (10, 11). The nucleotidyl attach- ment site is a lysine in the rat liver (13), reovirus (20), and Capping of eukaryotic pre-mRNA is the earliest detectable vaccinia virus guanylyltransferases (13, 21). The nucleotidyl- RNA processing event (1, 2). The cap consists of an inverted enzyme linkage is a phosphoamide bond between the cata- 7-methylguanosine residue linked via a 5'-5' triphosphate lytic lysine and GMP (10, 12-14, 21, 22), suggesting that the bridge to the first transcribed residue. Capping proceeds by the nucleotidyl transfer mechanism proceeds via nucleophilic sequential action of several enzymatic activities (reviewed in attack of the E-amino group of the lysine on the a phosphate refs. 3 and 4). Nascent RNA molecules synthesized by RNA of GTP (23). The extensive conservation of the capping polymerase II carry 5'-triphosphate termini. The capping en- reaction suggests that the GMP-lysine bond is a feature ofall zyme possesses a RNA 5'-triphosphatase activity that removes guanylyltransferases. the terminal phosphate and a guanylyltransferase (GTP:mRNA The S. cerevisiae capping enzyme is a heterodimer of guanylyltransferase, EC 2.7.7.50) activity that catalyzes the 80-kDa and 52-kDa subunits possessing 5'-triphosphatase transfer of a GMP moiety from GTP to the 5'-diphosphoryl and guanylyltransferase activities, respectively (24, 25). The group of the RNA. Subsequent methylation of the cap (5-7) essential gene CEGI encodes the small subunit (26) and is the may serve to prevent reversal ofthe guanylyl transfer reaction only cellular capping enzyme gene described to date. The (8). These reactions may be summarized as follows: gene for the large subunit has not yet been identified. The S. 'Yta RNA 5'-triphosphatase P i'+ (I) pppN- 0 ppN- + p y pca pax GTP:mRNA guanylyltransferase a PI'a' (II) pppG + ppN- w GpppN- + PPi X aPla' mRNA (guanine-7-)methyltransferase a3la' (III) GpppN- + AdoMet m7GpppNm- + AdoHcy. mRNA (ribose-2'-O-)methyltransferase The transfer ofGMP from GTP to the diphosphate termini of cerevisiae E-GMP linkage exhibits the characteristics of a mRNA (transguanylylation, reaction II) proceeds through phosphoamide bond (13)-i.e., alkali-stability, acid-lability, two reversible steps that involve an enzyme guanylylate and acidic hydroxylamine cleavage susceptibility (23). These (E-GMP) intermediate (9-11): properties are consistent with a GMP-lysine bond, although they could also be due to phosphoamide linkage to a histidine Yg~a a E + pppG E. pG + PPI or argiine. a a Abbreviations: ORF, open reading frame; WT, wild type; 5-FOA, E. pG + ppN- -. GpppN-+E 5-fluoroorotic acid; E-GMP, enzyme-GMP complex. *Present address: Department of Biological Chemistry and Molec- The publication costs ofthis article were defrayed in part by page charge ular Pharmacology, Harvard Medical School, 240 Longwood Av- payment. This article must therefore be hereby marked "advertisement" enue, Boston, MA 02115. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6624 Downloaded by guest on September 30, 2021 Biochemistry: Fresco and Buratowski Proc. Nadl. Acad. Sci. USA 91 (1994) 6625 In the course ofpolynucleotide ligation by DNA and RNA was isolated following infection with M13K07 helper phage. ligases, a covalent adenylyl-enzyme intermediate is formed. Mutagenic oligonucleotides were phosphorylated by using T4 AMP is attached to the enzyme by a phosphoamide bond with polynucleotide kinase and annealed to the phagemid DNA, a lysine that lies within a conserved motif (reviewed in ref. and extension and ligation reactions were performed as 27). The sequence surrounding the site ofGMP attachment in described (30). E. coli strain S28 (dut+ ung+) was trans- the vaccinia virus mRNA guanylyltransferase is similar to the formed with the reaction products to select against the motif containing the active-site lysine residues of DNA and uracil-containing parental strand. Plasmid DNA was isolated RNA ligases (16, 28). In contrast, the guanylyltransferase by alkaline lysis miniprep procedure, and mutants were active site from reovirus (20) does not obviously conform to identified by DNA sequencing. this consensus. While no extensive sequence similarities to The sequences ofthe mutagenic oligonucleotides, designed the viral capping enzymes are apparent, the guanylyltrans- to change the amino acids at Lys-59 and Lys-70 are as ferase of S. cerevisiae contains a short sequence that is follows: CEG1-K59mutS', 5'-CTGATGTGGAAGAGABBC- similar to the proposed consensus motif for nucleotidyl TGCTTGCGCATG-3', where B = T, C, or G; and CEG1- attachment (16, 28). K70mutS', 5'-GATTACTACGTTTGTGAGABNACAGA- To determine the guanylylated residue ofthe yeast capping TGGTCTGCGGG-3', where N = T, C, G, or A. enzyme, we performed phosphopeptide sequencing and ex- Yeast Methods. Plasmids were introduced into yeast by amined the effects ofreplacement ofthe suspected active-site using a modified lithium acetate transformation protocol (31). lysine. We report here that the site ofguanylyl attachment in Media preparation, tetrad dissection, genomic DNA isola- yeast CEG1 is Lys-70. The active-site sequence conforms to tion, plasmid shuffling, and all other yeast manipulations a motif for nucleotidyl attachment found in enzymes that were performed by standard methods (30,32). 5-Fluoroorotic catalyze nucleotidyl transfer to nucleic acid substrates. acid (5-FOA) was purchased from PCR Incorporated. Yeast Srains. Yeast strains were CTY143 (MATa/MATa ura3-52/ura3-52 leu2-3,112/leu2-3,112 his3A200/his3A20OX), MATERIALS AND METHODS YSB225 (MATa/MATa ura3-52/ura3-52 leu2-3,112/leu2-3, Plasmids. The CEGI gene was amplified from S. cerevisiae 112 his3A200/his3A200 CEGJ/cegll ::HIS3), YSB238 genomic DNA by the polymerase chain reaction (PCR) using (MATa/MATa ura3-52/ura3-52 leu2-3,112/leu2-3,112 his- oligonucleotide primers derived from the published genomic 3A2700/his3A200 CEGI/ceglAl::HIS3 {pRS316-CEG1}), and sequence (26). The entire CEGI gene was amplified by using YSB244, (MATa ura3-52 leu2-3,112 his3A2X00 ceglAl::HIS3 oligonucleotide primers CEG1-A5' and CEG1-C3'. The {pRS316-CEG1}). CEG1 open reading frame (ORF) was amplified by using CTY143 was transformed with Pst I/Sma I-digested primers CEG1-B25' and CEG1-C3', creating an Nco I site at pBSKS(+)(ARI-X)-ceglAl::HIS3. His+ transformants were the initiation codon in the process. The sequences of PCR selected after 3-6 days to generate YSB225. Disruption of primers are as follows: CEG1-A5', 5'-GGGAGATCTCAC- CEGI was verified by Southern analysis (data not shown). TGAAAGGCAGATGATC-3'; CEG1-B25', 5'-GGGAGAT- Sporulation resulted in 2:2 segregation of viability, and all CTGCCATGGTTTTAGCAATGGAAAGTAGAG-3'; and viable spores were His-. YSB225 was transformed with CEG1-C3', 5'-GGGAGATCTATAACACTTTCACCCTC- plasmid pRS316-CEG1 to create YSB238. A Ura+ His+ GCTTCTC-3'. haploid strain (YSB244) derived from YSB238 was used for The PCR product containing the entire CEGI gene was plasmid shuffling experiments. digested with Bgl II and ligated into the BamHI site of Prifcation of Recombinant Guanylyltransferase. Polyhis- pRS315, pRS316 (29), or pBSKS(+), generating pRS315- tidine-tagged CEG1 (His7-CEG1) proteins were expressed by CEG1, pRS316-CEG1, and pBSKS(+)-CEG1, respectively. using a 17 promoter/polymerase system in E. coli strain The PCR product containing the CEGI ORF was digested pGP1-2/K38 (33). Strains carrying bacterial expression plas- with Bgl II and inserted into the BamHI site ofpBSKS(+) to mids were grown at 300C to an OD595 =0.5 and induced by create plasmid pBSKS(+)-CEGlorf. warming the cultures to 420C for 15 min. Cells were incubated For bacterial production of CEG1 protein and to facilitate at 3TC and harvested 3 hr after induction. Lysates were purification by Ni2+ chelate chromatography, the ORF was prepared, and soluble recombinant proteins were purified by cloned downstream of a phage 17 promoter and engineered chromatography over a nitrilotriacetic acid-agarose column to carry seven histidine residues at the N terminus.
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