Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.
A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol 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 themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.
Neufeld
K L
KL
Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Galarza
J M
JM
Richards
O C
OC
Summers
D F
DF
Ehrenfeld
E
E
D001483:Base Sequence; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D017955:Poliovirus / Q000201:enzymology*; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity; D014779:Virus Replication
D009841:Oligonucleotides; D012367:RNA, Viral; D009713:Nucleotidyltransferases; C020658:glutamine-synthetase adenylyltransferase; D012324:RNA-Dependent RNA Polymerase