Transfer Of GMP Research Articles

Mutational analysis of yeast mRNA capping enzyme.

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases.

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.

Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli.

The D1 gene encoding the large subunit of vaccinia virus mRNA capping enzyme was expressed in Escherichia coli BL21(DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. Two major species of protein-GMP complex were formed, one of Mr 95,000 (corresponding in size to the D1 gene product) and one of Mr 60,000. Partial purification of the guanylyltransferase was effected by ammonium sulfate precipitation and ion-exchange chromatography. The expressed large subunit synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor, but was unable to catalyze cap methylation in the presence of S-adenosylmethionine. Thus, the small capping enzyme subunit was shown to be dispensable for guanylylation, but required for cap methylation of RNA. The Mr 95,000 and Mr 60,000 protein-GMP forming activities were resolved during centrifugation in a glycerol gradient; the two forms sedimented at 5.5 S and 4.4 S, respectively, consistent with each enzyme form being a monomer. Either species catalyzed GMP transfer to an RNA acceptor. The isolated Mr 95,000 guanylyltransferase could be converted to an active Mr 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the Mr 95,000 polypeptide.

RNA capping by the vaccinia virus guanylyltransferase. Structure of enzyme-guanylate intermediate.

GTP:RNA guanylyltransferase isolated from vaccinia virus catalyzes the transfer of GMP from GTP to the 5' terminus of RNA via an enzyme-guanylate intermediate. Incubation of the purified vaccinia RNA guanylyltransferase with [alpha- 32P]GTP and MgCl2 yields [32P]GMP covalently linked to the Mr = 95,000 subunit. The bond involves the phosphate moiety of GMP and the Ne-amino group of lysine. This was verified by treatment of the isolated 95-kDa subunit-[32P]GMP complex with sodium periodate, followed by methylamine-catalyzed beta-elimination. The product was then hydrolyzed by alkali producing 32P-labeled lysine (Ne-P)phosphate.

Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.

Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5'-triphosphate or diphosphate terminus of RNA to generate the cap structure G(5')ppp(5')N-. The guanylylation reaction consists of a series of at least two partial reactions: (i) GTP + E in equilibrium E-pG + PPi, (ii) E-pG + (p)ppNpNpN- leads to GpppNpNpN- + E. Inthe first of these, GTP reacts with capping enzyme in the absence of an RNA acceptor to form a covalent enzyme-guanylate intermediate. The GMP is linked to the Mr 95,000 subunit of the capping enzyme via a phosphoamide bond, as judged by the acid-labile, alkali-stable nature of the bond and by the susceptibility of the linkage to cleavage by hydroxylamine at pH 4.75. The isolated enzyme-guanylate complex is able to transfer the guanylate moiety to triphosphate-terminated poly(A) to yield the 5' cap structure GpppA or to pyrophosphate to regenerate GTP. Both partial reactions of transguanylylation require a divalent cation.