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 synt hesis 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 d omains involved in cap formation have been assigned to individual subu nits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are require d for transcription termination. The 844-amino acid D1 subunit by itse lf; which is fully active in triphosphatase and guanylyltransferase fu nctions, has no demonstrable VTF activity in vitro. Neither does the D 12 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 format ion or by a H682A/Y683A double mutation of the D1 subunit, which abrog ates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and meth yl transfer.