Transcription initiation factor activity of vaccinia virus capping enzyme is independent of mRNA guanylylation

Abstract

Cytoplasmic extracts of vaccinia virus-infected HeLa cells blocked in DNA replication were capable of transcribing templates containing the minimal promoter sequences derived from three viral intermediate-stage genes (A1L, A2L, and G8R) but not promoters from early or late genes. One of three isolated components required for transcription copurified with the viral capping enzyme, a heterodimeric protein responsible for forming the 7-methyl-guanosine(5')triphospho(5')nucleoside [m7G(5')ppp(5')N-] structure at the 5' end of mRNAs, as had been reported using a template with another intermediate promoter [Vos, J. C., Sasker, M. & Stunnenberg, H. G. (1991) EMBO J. 10, 2553-2558]. Transcription factor activity was associated with partially, purified capping enzyme from infected cell extracts, homogeneous enzyme from purified virions, and recombinant viral enzyme from Escherichia coli. By transcribing truncated templates of different sizes, we determined that RNA chains of 35 nt were capped whereas those of 15 nt were not. Nevertheless, the capping enzyme was required for formation of short uncapped transcripts, indicating that capping and transcription initiation factor activities are independent functions.