Purification of wheat germ RNA ligase. I. Characterization of a ligase-associated 5'-hydroxyl polynucleotide kinase activity.
An RNA ligase that catalyzes the formation of a 2'-phosphomonoester-3',5'-phosphodiester bond in the presence of ATP and Mg2+ was purified approximately 6000-fold from raw wheat germ. A 5'-hydroxyl polynucleotide kinase activity copurified with RNA ligase through all chromatographic steps. Both activities cosedimented upon glycerol gradient centrifugation even in the presence of high salt and urea. RNA ligase and kinase activities sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. The purified polynucleotide kinase activity required dithiothreitol and a divalent cation for activity and was inhibited by pyrophosphate and by ADP. The kinase phosphorylated a variety of 5'-hydroxyl-terminated polynucleotide chains including some that were substrates for the RNA ligase (e.g. 2',3'-cyclic phosphate-terminated poly(A)) and others that were not ligase substrates (e.g. DNA or RNA containing 3'-hydroxyl termini). RNA molecules containing either 5'-hydroxyl or 5'-phosphate and 2',3'-cyclic or 2'-phosphate termini were substrates for the purified RNA ligase activity. The rate of ligation of 5'-hydroxyl-terminated RNA chains was greater than that of 5'-phosphate-terminated molecules, suggesting that an interaction between the wheat germ kinase and ligase activities occurs during the course of ligation.
D000838:Anions; D002413:Cations, Divalent; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000302:isolation & purification* / Q000378:metabolism; D011118:Polynucleotide Ligases / Q000302:isolation & purification*; D011130:RNA Ligase (ATP) / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology
D000838:Anions; D002413:Cations, Divalent; D009097:Multienzyme Complexes; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)