pyrophosphate and 8-azidoadenosine-5--triphosphate

Avian reovirus S1133 was shown to contain all the enzymatic activities required for the synthesis of mature viral transcripts, including a dsRNA-dependent RNA polymerase, a nucleoside triphosphate phosphohydrolase, an mRNA guanylyltransferase, and two mRNA methyltransferases. The virus used these enzymes both in vitro and in vivo to catalyze the synthesis of viral mRNAs containing a type-1 cap at their 5' ends. Incubation of reovirions with GTP led to the formation of an intermediate structure consisting of GMP bound to the viral core protein lambda 3 through a phosphoamide linkage. The reaction was specific for GTP and required the presence of both Mg2+ and inorganic pyrophosphatase. The GMP moiety can be transferred from the lambda 3-GMP complex to acceptors such as GDP and GTP, yielding GpppG and GppppG, respectively. Our results demonstrate that lambda 3 is the avian reovirus guanylyltransferase.

Topics: Adenosine Triphosphate; Animals; Azides; Cells, Cultured; Chick Embryo; Cyclic GMP; Diphosphates; DNA-Directed RNA Polymerases; Fibroblasts; Kinetics; Nucleotidyltransferases; Reoviridae; Ribonucleotides; RNA, Messenger; RNA, Viral; Transcription, Genetic

The photoaffinity probes [gamma-32P]2-azidoATP (2-N3ATP) and [alpha-32P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.J., Karikó, K., Sobol, R.W., Jr., Li, S.W., Reichenbach, N.L., & Haley, B.E., preceding paper]. In this study we show that 2- and 8-N3ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N3ATP and 8-N3ATP into the enzyme. The covalent photoinsertion of [alpha-32P]8-N3ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling of 2-5A synthetase is saturated at 1.5 mM 2-N3ATP and 2.0 mM 8-N3ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggests the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N3ATP into the binding site(s) of the synthetase was as follows: ATP greater than 2'dATP = 3'dATP greater than CTP greater than ITP greater than AMP greater than NAD+ greater than UTP greater than UMP greater than CMP. Photoinsertion of 8-N3ATP into 2-5A synthetase increases with the addition of poly(rI).poly(rC).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2',5'-Oligoadenylate Synthetase; Adenosine Triphosphate; Affinity Labels; Azides; Binding, Competitive; Diphosphates; Kinetics; Phosphorus Radioisotopes; Photochemistry; Ultraviolet Rays