IDENTIFICATION OF TERMINAL ADENYLYL TRANSFERASE-ACTIVITY OF THE POLIOVIRUS POLYMERASE 3D(POL)

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3 D(pol)). Highly purified 3D(pol) is capable of adding [P-32]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The p urified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-poly acrylamide gel electrophoresis and renatured, retained the TATase acti vity. Two 3D(pol) mutants, purified from Escherichia coli expression s ystems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. col i strain that harbored no expression plasmid were unable to catalyze f ormation of the TATase products. With the RNA oligonucleotide 5'-CCUGC UUUUGCA-3' used as an acceptor, the products formed by wild-type 3D(po l) were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA,either by themsel ves or when all deoxynucleoside triphosphates were present in the reac tion. Results from kinetic and stoichiometric analyses suggest that th e reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an ac ceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilizatio n and the nature of the products formed during the reaction were depen dent on the acceptor RNA.