Abstract
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.
Highlights
An RNA ligase that catalyzes the formation of a2’- half
0.24 11,4050,20810,8070,0500 rification of RNA ligaseD. uring the course of the purification, RNA ligase was assayed and fractions were pooled . 5'-Hydroxyl polynucleotide kinase activity was monitored at each step
The ligase activity sedimented as a single peakon glycerol gradients with a sedimentation coefficient of 6.2 S, corresponding to a protein doublet of about 110 kDa that represented approximately 70% of the protein detected by silver staining of polyacrylamide gels
Summary
Nuclease that accurately cleaves precursor tRNA to generate Since wheat germRNAligasewas active on synthetic the cognate halves This tRNA endonuclease (4)is a mem- substrates that could beeasily prepared and since wheat germ brane-associated enzyme that cleaves pre-tRNA molecules in could be obtained in large quantities, it was an attractive the absence of ATP to leave 2‘,3’-cyclic phosphate termini choice for the preparation of a eukaryotic RNA ligase that on the 5’-tRNA half and 5’-hydroxyl termini on the 3’-tRNA couldbe subjected to biochemical analysis. Who used tRNA halves generated by the yeast tRNA endonuclease containing 5’-hydroxyl and 2’,3‘-cyclic phosphate termini as substrate (7) In this case, a 2’,3’-cyclic phosphodiesterase as well as a 5’-hydroxylpolynucleotidekinase activity was associated withthe RNA ligase.
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