Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.
Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.
D002467:Cell Nucleus / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*; D012315:RNA Caps / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism*; D013696:Temperature
D012315:RNA Caps; D012333:RNA, Messenger; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase