Purification of RNA 3'-terminal phosphate cyclase from HeLa cells. Covalent modification of the enzyme with different nucleotides.
RNA 3'-terminal phosphate cyclase has been purified about 6000-fold to near homogeneity from HeLa cells. The purified protein is a single polypeptide with an Mr of 38,000-40,000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0-9.0. In the presence of Mg2+ and ATP this enzyme catalyzes the conversion of a 3'-phosphate group into the cyclic 2',3'-phosphodiester at the 3' end of RNA, through formation of a covalent cyclase-AMP intermediate. GTP, CTP and UTP (but not dATP or ADP) can also function as cofactors in the cyclization reaction, although less efficiently (apparent Km values for ATP and GTP are 6 microM and 200 microM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [alpha-32P]NTPs.
Vicente
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Friedrich Miescher-Institut, Basel, Switzerland.
Filipowicz
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology*; D014544:Uridine Triphosphate / Q000494:pharmacology
D009711:Nucleotides; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008025:Ligases; C037451:RNA 3'-terminal phosphate cyclase; D014544:Uridine Triphosphate