Characteristics of reactions catalyzed by purified guanylyltransferase from vaccinia virus.

Abstract

<‘” GTP:RNA guanylyltransferase catalyzes the initial reaction in the formation of “caps” at 5’ termini of mRNAs. The characteristics of the guanylation reaction have been studied using purified enzyme isolated from vaccinia virus. Guanylation requires a divalent cation (Mg2’), is stimulated by monovalent cations (Na+ and K+), and is unaffected by S-adenosylmethionine. The enzyme utilizes GTP as guanylyl donor with an apparent K, of 15 PM; it will guanylate a variety of RNAs. The enzyme specifically requires V-triphosphate-terminated RNA chains, as shown from studies with poly(A) containing 5’-triphosphate, 5’-diphosphate, 5’monophosphate, or 5’-hydroxyl termini; and from studies with the dinucleotides pppGpC and ppGpC. The apparent K, for termini of 5’4riphosphate poly(A) is 0.2 PM. The triphosphate bridge of cap structures synthesized by purified guanylyltransferase is derived from the a-phosphate of GTP, and the a- and /3-phosphates of the triphosphate-terminated RNA acceptor. During guanylation of 5’-triphosphate poly(A), Pi and PPi are each released in a 1: 1 stoichiometry with “cap” formation; Pi and PPi are derived from B’-triphosphateterminated poly(A) and GTP, respectively. The results indicate that the reaction catalyzed by purified guanylyltransferase with GTP and 5’-triphosphate-terminated poly(A) is: uPa 1/P’a’ ap’a’ 1/ UP