Abstract
The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.
Highlights
The enzyme, RNA cyclase, has been purified from scribed as collinear precursor molecules from whicthhe intercell-free extracts of HeLa cells approximately 6000- vening sequences are excised by a cleavage-ligation reaction, fold
The 5‘-hydroxyl group is phosphorylated by transfer of the y-phosphate from ATP, and it is hydroxylpolynucleotide kinase, RNA cyclase, and thought that thisend is furtheractivated by transfer of AMP
Filipowicz et al [23] discovered that HeLa cell extracts con- were run inthe same lanes, and theirpositions are noted in the center tain an activity that converts 3”phosphate terminated RNA chains to the2’,3’-cyclic phosphate derivative
Summary
The enzyme, RNA cyclase, has been purified from scribed as collinear precursor molecules from whicthhe intercell-free extracts of HeLa cells approximately 6000- vening sequences are excised by a cleavage-ligation reaction, fold. The heparin-Ultrogel fractions contained an activity that hydrolyzed small amounts of ATP and ATPyS toAMP and PPi or thiopyrophosphate in the absence of added 3"phosphate terminated RNA under the conditions specified in Fig. IOA, lane 4 (see Table IV).
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