Structure of vaccinia virus late promoters

Functional elements of vaccinia virus late promoters were characterized by mutagenesis. Synthetic oligonucleotides 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 122 recombinants thus obtained was assayed for β-galactosidase expression. The relative amounts and 5′ ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension. The analysis indicated that late promoters may be considered in terms of three regions; an upstream sequence of about 20 base-pairs, rich in T and A residues, separated by a spacer region of about six base-pairs from a highly conserved (−1)TAAAT(+4) element within which transcription initiates. All single nucleotide substitutions within the three A residues of the TAAAT, as well as the addition of a fourth A residue, caused drastic reductions in promoter strength. All substitutions of the T residues at −1 and +4 were also detrimental to promoter activity, to an extent that depended on the strength of the promoter as determined by the upstream sequence. mRNA synthesis appeared to initiate within the three A residues regardless of promoter strength. The 5′-poly(A) leader, which is a unique feature of poxvirus late mRNAs, was diminished in length when either of the T residues at −1 and +4 was mutated, was absent or limited to a few nucleotides when any of the three A residues was substituted, but was unaffected by changes outside the TAAAT sequence. The data are consistent with a model for the generation of the normal 5′-poly(A) leader by an RNA polymerase slippage mechanism requiring three consecutive A residues. Single nucleotide substitutions within the six base-pairs upstream and three base-pairs downstream from the TAAAT sequence had modest effects on promoter strength. The most and least favourable changes led to a fourfold increase and an eightfold decrease in activity, respectively. Sequences further upstream were essential for late promoter function; tracts of T or A residues enhanced expression up to 20-fold, the former conferring much greater activity. Highest expression was obtained with a tract of 18 or 20 T residues. Information gained from the mutational analysis allowed the construction of a synthetic late promoter that is 100-fold stronger than the late promoter used initially, and over 50% stronger than copies of the most efficient known vaccinia and cowpox virus natural late promoters. The synthetic promoter was incorporated into new recombination vectors designed for high-level expression of foreign genes in vaccinia virus.