All 4 bases of both strands at -9 and -8 in T7 promoter are needed to be substituted by SP6-specific bases to switch promoter specificity.

It has previously been shown that a substitution of two base pairs at -9 and -8 in T7 promoter by SP6 promoter-specific base pairs alone is sufficient to make the T7 variant become an SP6 promoter, abolishing T7 promoter activity [Lee, S. S., and Kang, C. (1992) Biochem. Int. 26, 1-5]. In order to determine which of the 4 bases on both strands at the two positions is involved in the discriminative specificity, each base or each pair of bases was substituted by SP6-specific base(s), allowing one or two mismatches. The results show that all-four-base substitution only is capable of switching promoter specificity, and that all the four bases are substantially involved in T7 promoter activity.