The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme.
Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.
Luo
Y
Y
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Mao
X
X
Deng
L
L
Cong
P
P
Shuman
S
S
D001483:Base Sequence; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D013728:Terminator Regions, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins
D009097:Multienzyme Complexes; D012367:RNA, Viral; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus