Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.
Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.
Yu
L
L
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
S
S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D006157:Guanosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014616:Vaccinia virus / Q000201:enzymology*; D000098556:mRNA Guanylyltransferases
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006157:Guanosine Monophosphate; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; C023090:RNA triphosphatase; D000409:Alanine