Purification of RNA 3′‐terminal‐phosphate cyclase from HeLa cells

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Abstract
RNA S′‐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 38000‐40000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0‐9.0. In the presence of Mg 2+ 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 μM and 200 μM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [α‐32P]NTPs.