Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme

Abstract

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 ϳ1% of wild-type specific activity) suggests that both ␥-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 ␥-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 ␥-phosphate cleavage is rate limiting.

RNA methyltransferases involved in 5′ cap biosynthesis

“…The D1 subunit contains triphosphatase and guanylyltransferase activities in the N-terminal domain, and a cap0 methyltransferase domain that forms a heterodimer with the D12 subunit. 28,29 The methyltransferase active site is located entirely in the D1 subunit and has a weak cap0 modification activity that is stimulated allosterically by D12. [30][31][32] Interestingly, the D12 structure resembles a degenerate cap 2 0 -O-ribose methyltransferase domain (see below), but it lacks a proper SAM binding site and does not show any methyltransferase activity on its own.…”

Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme

“…Catalytic domains are organized in a modular fashion. The amino-terminal 545-amino-acid segment of the 844-amino-acid D1 subunit catalyzes the triphosphatase and guanylyltransferase reactions (9,16,29,34). The methyltransferase reaction is catalyzed by a distinct, nonoverlapping domain consisting of the carboxyl portion of the D1 subunit heterodimerized with the D12 protein (1,10,13).…”

“…Vaccinia virus capping enzyme hydrolyzes the ␥-phosphate of triphosphate-terminated RNA and the ␥-phosphate of nucleoside triphosphates (NTPs) (31,33). Both reactions occur at a single active site, which is distinct from the active site of the guanylyltransferase (17,34). The triphosphatase active site includes essential constituents located within the N-terminal half of the D1(1-545) protein.…”

“…We characterized two alanine cluster mutations within this region, R77 to A (R77A)-K79A and E192A-E194A, that inactivate the triphosphatase but not the guanylyltransferase (34). A novel property of the R77A-K79A and E192A-E194A proteins was their ability to catalyze GMP transfer to a virgin triphosphate end of RNA to generate the unusual dinucleoside tetraphosphate cap, GppppA (34).…”

“…The individual catalytic contributions of Arg-77, Lys-79, were gauged by replacing each residue by alanine. The mutations were introduced into the D1 (1-545) gene via the two-stage PCR overlap extension strategy (34). NdeI-BglII restriction fragments of the PCR-amplified mutated D1(1-545) DNAs were inserted into pET16b, thereby generating a series of pET-His-D1(1-545)-Ala expression plas-mids.…”