A ROLE FOR THE H4 SUBUNIT OF VACCINIA RNA-POLYMERASE IN TRANSCRIPTIONINITIATION AT A VIRAL EARLY PROMOTER

The vaccinia virus H4 gene encodes an essential subunit of the DNA-dep endent RNA polymerase holoenzyme encapsidated within virus particles ( Ahn, B., and Moss, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3536- 3540; Kane, E. M., and Shuman, S. (1992) J. Virol. 66, 5752-5762). The role of this protein in transcription of viral early genes was reveal ed by the effects of affinity-purified anti-H4 antibody on discrete ph ases of the early transcription reaction in vitro. Anti-H4 specificall y prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary com plexes. Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa su bunit of the vaccinia early transcription initiation factor (ETF). Nat ive gel mobility-shift assays showed that anti-H4 prevented the NTP-de pendent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter. Two species of ternary complexes coul d be resolved by native gel electrophoresis. Addition of anti-H4 to pr eformed complexes elicited a supershift of both ternary species but no t of the preinitiation complex. Supershift by anti-D6 revealed that th e more rapidly migrating species of ternary complex did not contain im munoreactive ETF. Loss of ETF from the ternary complex was time-depend ent. Thus, whereas the H4 protein was a stable constituent of the elon gation complex, ETF was dissociable. We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of ea rly promoters by the core RNA polymerase. H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation co mplex is established.