Early promoter-binding factor from vaccinia virions.

A factor, present in transcriptionally active extracts prepared from purified vaccinia virus particles, binds to vaccinia early promoter sequences. The specificity of binding was demonstrated by electrophoretic mobility shift assays using the 5'-terminal segments of two early genes and related and unrelated competitor DNA fragments. DNase I "footprint" analysis indicated that the factor formed a complex with promoter regions of both genes and protected sequences of 10-15 nucleotides centered 21-24 nucleotides upstream of the RNA start sites. The lack of protection of a late regulatory sequence and of an early promoter with transcriptionally inactivating single-nucleotide substitutions suggested that the protein is an early transcription factor. When subjected to glycerol gradient centrifugation, the DNA-binding factor was resolved from RNA polymerase and sedimented as a 7.5S species with an estimated molecular weight of 130,000.