ENZYMATIC REACTIONS AT THE TERMINI OF RIBONUCLEIC ACID (RNA LIGASE, SPLICING, PROCESSING, CYCLIC PHOSPHATE TERMINI, 5'-HYDROXYL POLYNUCLEOTIDE KINASE)
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Wheat germ contains an RNA ligase activity that catalyzes the formation of an unusual 2'-phosphomonoester,3'5'-phosphodiester bond, in the presence at ATP and Mg('++). A procedure was developed that resulted in a 6000-fold purification of this enzyme from raw wheat germ. Purified RNA ligase sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. 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. Purified RNA ligase preparations also contained a 2',3'-cyclic phosphodiesterase activity that hydrolyzed 2',3'-cyclic phosphate termini to yield 2'-phosphate termini, in the absence or presence of ATP. All three activities interacted during the ligation of RNA molecules containing 5'-hydroxyl and 2',3'-cyclic phosphate termini, as follows. The phosphate in the 2',3'-cyclic bond was shifted to the 2'-position by the cyclic phosphodiesterase activity, leaving a free 3'-hydroxyl group. A phosphate from the (gamma)-position of ATP was transferred to the 5'-hydroxyl terminus of the RNA by the kinase activity. This newly generated 5'-phosphate terminus was then joined to the available 3'-hydroxyl terminus by the RNA ligase activity, to form a new 3',5'-phosphodiester bond. Ligation of one mole of 5'-P, 2',3'-cyclic P terminated poly(A) was accompanied by the hydrolysis of one mole of ATP to one mole each of AMP and PP(,i). Purified RNA ligase catalyzed an ATP-PP(,i) exchange reaction and formed a covalent enzyme-adenylate (E-AMP) complex. The unique feature of these reactions was their marked stimulation (up to 400-fold) by the addition of RNA.;Extending my studies to mammalian systems, a search was made for RNA ligase activities in various types of preparations of HeLa cells. An enzymatic activity was identified in a cytoplasmic fraction that adenylylated the 5'-P terminus of poly(A) chains.