Structure of vaccinia virus early promoters

Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression. Synthetic oligonucleotides, based primarily on the sequence of the 7.5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus. The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for beta-galactosidase expression. The relative amounts and precise 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. Many promoters were tested for their ability to direct specific transcription in vitro. A generally good correlation was noted between measurements of promoter strength estimated by beta-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro. A relatively simple picture emerged from the analysis. The early promoter consists of a 16 base-pair critical region, in which most single nucleotide substitutions have a major effect on expression, separated by 11 base-pairs of a less critical T-rich sequence from a seven base-pair region within which initiation with a purine usually occurs. For the critical region of the 7.5-kD promoter, AAAAgTaGAAAataTA, any substitution of an upper-case nucleotide reduced expression, usually drastically, whereas certain substitutions of lower-case nucleotides maintained or significantly enhanced expression. On the basis of this analysis, the wide range of activities of natural promoters could be attributed to the presence of one or more non-optimal nucleotides in the critical region. Moreover, single nucleotide substitutions in such promoters had the predicted enhancing effects. Most mutations in the critical region of the 7.5-kD promoter behaved independently, but some nucleotide substitutions compensated for potentially detrimental nucleotides at other positions. Promoters substantially stronger than any natural ones examined were constructed by combining several up-mutations within the critical region of the 7.5-kD promoter and by repeating the critical region sequence. Like the TATA box of eukaryotic RNA polymerase II promoters, the critical region specifies the site of transcriptional initiation.