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Conserved in Schizosaccharomyces Pombe and Viral Capping Enzymes

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Conserved in Schizosaccharomyces Pombe and Viral Capping Enzymes Proc. Nati. Acad. Sci. USA Vol. 91, pp. 12046-12050, December 1994 Biochemistry Covalent catalysis in nucleotidyl transfer reactions: Essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases STEWART SHUMAN*, YIZHI Liut, AND BEATE SCHWERt *Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021; and tDepartment of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854 Communicated by Bernard Moss, August 1, 1994 ABSTRACT Formation of the 5' cap structure of eukary- based comparisons of the yeast and viral proteins uncovered otic mRNAs occurs via transfer of GMP from GTP to the 5' no conservation of primary sequence. However, mapping of terminus of the primary transcript. RNA guanylyltansferase, the yeast guanylyltransferase active site to Lys-70 within the the enzyme that catalyzes this reaction, has been isolated from motifKTDG (11, 12), which is identical to the KTDG element many viral and cellular sources. Though differing in molecular at the vaccinia active site (7, 8), argues that the cellular and weight and subunit structure, the various guanylyltranferases DNA virus-encoded enzymes might have a common evolu- employ a common catalytic mechanism involving a covalent tionary origin. enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae Covalent catalysis during nucleotidyl transfer is not unique CEGI is the sole example ofa cellular capping enzyme gene. In to the RNA capping reaction. DNA and RNA ligation reac- this report, we describe the Identification and characterization tions were the first examples of nucleotidyltransferases that of the PCEI gene encoding the capping enzyme from Schizo- employ an enzyme-adenylate intermediate (13). The AMP saccharomycespombe. PCEI was isolated from a cDNA library moiety is linked covalently via a phosphoamide bond to a Lys by functional complementation in Sa. cerevisiae. Induced ex- residue of the ligase polypeptide (14). The bound AMP is pression of PCEI in bacteria and in yeast confirmed that the transferred to the 5' monophosphate end of a polynucleotide 47-kDa Sc. pombe protein was enzymatically active. The amino toformanactivatednucleicacidintermediate-A(5')pp(5')N- acid sequence ofPCE1 is 38% identical (152 of 402 residues) to that is reminiscent ofthe G(5')ppp(5')N RNA cap. Mapping of the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the active sites of DNA and RNA ligases to Lys residues the two cellular capping enzymes with guanylyltranferases within KXDG motifs (15, 16) suggests that capping enzymes encoded by DNA viruses revealed local sequence similarity at and polynucleotide ligases may be related structurally and the enzyme's active site and at four additional collinear motifs. functionally. Mutational analysis of yeast CEGI demonstrated that four of To gain further insight into the mechanism and molecular the five conserved motifs are essential for capping enzyme evolution of nucleotidyltransferases, we have identified and function in vivo. Remarkably, the same motifs are conserved in characterized a cDNA encoding the mRNA capping enzyme the polynucleotide ligase family of enzymes that employ an from the fission yeast Schizosaccharomyces pombe.t The enzyme-(Lys-AMP) intermediate. These findings illuminate a Sc. pombe PCE1 gene encodes a 402-aa polypeptide with shared structural basis for covalent catalysis in nucleotidyl extensive sequence identity to the guanylyltransferase from transfer and suggest a common evolutionary origin for capping Sa. cerevisiae. The cellular enzymes and the DNA virus enzymes and ligases. guanylyltransferases display local sequence similarity at the active site and at four additional collinear motifs not appre- RNA guanylyltransferase (capping enzyme) catalyzes transfer ciated previously. Remarkably, the same five motifs are ofGMPfrom GTP to the 5' end ofmRNA. The hallmark ofthe conserved in the polynucleotide ligase family of enzymes. RNA capping reaction is the formation of an enzyme- Mutational analysis demonstrates that the conserved motifs guanylate intermediate in which GMP is linked covalently to are essential for capping enzyme function. a Lys residue of the protein via a phosphoamide bond (1). Analysis ofvirus-encoded RNA guanylyltransferases points to at least two families of capping enzymes, based on overall MATERIALS AND METHODS sequence conservation and on the nature of the enzyme's cDNA Encoding Sc. pombe Capping Enzyme-Cloning by active site (2-8). The capping enzymes encoded by double- Genetic Complementation. The galactose-dependent yeast strand DNA viruses (vaccinia, Shope fibroma, and African strain YBS3 [MATa, leu2, lys2, trpl, ura3, cegl::hisG, swine fever) display local sequence similarities that suggest a pGAL-CEG1 (CEN, TRPI, GALIO-CEGI)] was used to common evolutionary origin-this includes a conserved screen a Sc. pombe cDNA library in the vector pDB20 (2 ,nm, KXDG motif at the site of covalent guanylylation (5, 6). URA3) (17). Ura+ transformants were selected at 300C on Guanylyltransferase encoded by reovirus, a double-strand medium containing glucose. Plasmids with cDNA inserts RNA virus, displays no obvious sequence similarity to the were recovered from viable yeast colonies, amplified by vaccinia protein despite their essentially identical enzymatic transformation in Escherichia coli, and then retested for functions (7, 8). Although guanylyltransferases have been complementation of YBS3 growth on glucose. All secondary isolated from numerous cellular sources (9), a cellular gene transformants were incapable of growth on glucose in the (CEGI) encoding an RNA capping enzyme has only been presence of 5-fluoroorotic acid (5-FOA), indicating that the identified in Saccharomyces cerevisiae (10). Initial computer- growth on glucose required the library plasmid. DNA pre- The publication costs ofthis article were defrayed in part by page charge Abbreviation: 5-FOA, 5-fluoroorotic acid. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U16143). 12046 Downloaded by guest on September 28, 2021 Biochemistry: Shuman et al. Proc. Nadl. Acad. Sci. USA 91 (1994) 12047 pared from five secondary colonies was amplified in bacteria CTCTAAAGAAGCCCTCTCTTAGCAAAGAAACGAGTGTATTATTAAAAGGA ATG GCA CCC TCA GAG AAA GAC ATT GAA GAG GTA TCA GTC CCT GGA GTT TTA GCA CCG CGC GAC GAT and screened by restriction digestion with EcoRI, HindIII, MA P S E K D I E E V S V P G V L A P R D D andBamHI. All five plasmids contained inserts with identical GTG AGG GTT TTA AAG ACA CGA ATT GCC AAA TTA TTA GGA ACA AGT CCT GAT ACA TTT CCT GGA TCA restriction patterns. HindIll and EcoRI fragments from one V RV L K T R I A K L L G TS PDT F P G S CAG CCA GTT TCT TTT TCA AAG AAA CAT TTA CAA GCA TTA AAA GAA AAG AAC TAT TTC GTA TGT GAA cDNA clone were inserted into pBS-KS+ (Stratagene). The Q P V S F S K K H L Q A L K E K NYF V C E AAA AGT GAT GGA ATT CGT TGT TTA CTT TAT ATG ACC GAG CAT CCT CGG TAC GAA AAT CGA CCC AGT nucleotide sequence of the inserts was determined by dide- K S D G I R C L L Y M T E H P R Y E N R P S oxynucleotide sequencing. Sequencing of overlapping re- GTA TAT TTA TTT GAT CGT AAA ATG AAT TTT TAT CAT GTT GAG AAA ATT TTT TAT CCA GTT GMA AAT striction fragments and of the intact cDNA clone made clear V Y L FD R K M NF Y H V e K I F Y P V E N GAC AAA TCT GGA AAA AAA TAT CAT GTT GAT ACA CTT TTG GAC GGT GAG TTG GTT TTA GAT ATC TAT the order of the fragments and the 5' and 3' margins of the DK S G K KY H V D T L L D G E L V L D I Y cDNA insert. The cloned Sc. pombe gene was designated CCA GGT GGT AAG AAG CAA CTG AGA TAT TTA GTC TTT GAT TGT TTG GCA TGT GAT GGA ATT GTT TAT PCEI. P GG KK Q L R Y L V F D C L A C D G I V Y ATG AGT CGA TTG CTT GAC AAA CGC TTG GGA ATT TTT GCT AAA AGC ATT CAA AAG CCC TTA GAT GAA Vectors for Expression of Sc. pombe Capping Enzyme. The N S R LL D K R L G I F A K S I Q K P L D E PCEI coding sequence was isolated from the cDNA insert TAT ACA AAG ACT CAT ATG CGC GAA ACT GCC ATA TTT CCT TTT CTC ACA TCG TTA AAA AAA ATG GAG by Y T K T H H R E T A I F P F L T SL K K M E PCR amplification using oligonucleotides corresponding to CTG GGT CAT GGT ATC CTA AAG TTA TTT AAT GAA GTG ATC CCC CGA CTT CGT CAT GGT AAT GAT GGA the 5' end of the open reading frame and the 3' untranslated L G H G I L K L F N E V I P R L R H G N DG CTT ATC TTT ACA TGT ACG GAA ACT CCT TAT GTA TCT GGC ACT GAC CAG TCG CTT TTG MG TGG AAA region. The 5' oligonucleotide introduced an Nco I restriction L I F TCT E T P Y V S G TD Q S L L K H K site at the start codon and the 3' oligonucleotide included a CCA AM GAA ATG AAT ACA ATA GAC TTT ATG CTA AAG CTG GAA TTT GCA CAG CCT GM GAA GGG GAC BamHI site. The amplified DNA fragment was digested with P K E M NT I D F M L K L E F A Q P E E G D ATT GAT TAT TCA GCC ATG CCA GAA TTT CAA CTT GGT GTA TGG GAG GGT AGG AAC ATG TAC TCT TTT Nco I and Bgl II (which cuts at a single site in the cDNA I D Y S A N P E F Q L G V W E G R N M Y S F the and Tm GCC TTC ATG TAT GTT GAT GAA AAA GAA TGG GAA AAA TTG AAA AGC TTT AAT GT? CCT TTA TCG immediately downstream of translation stop codon) FAFM Y V D E K E WE K L K S F N V P L S then ligated into the bacterial expression vector pET14b.
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