2 BASE-PAIRS AT -9 AND -8 DISTINGUISH BETWEEN THE BACTERIOPHAGE-T7 AND SP6-PROMOTERS

Bacteriophage T7 and SP6 RNA polymerases and their promoters share a h igh degree of their primary structure homology, but each polymerase ex clusively recognizes its own promoter sequence. To reveal the molecula r basis of this specificity, 4 base pairs at positions -12, -10, -9, a nd -8 of the T7 promoter were substituted individually and multiply by SP6 promoter-specific base pairs, and 3 base pairs at -10, -9, and -8 of the SP6 promoter were replaced by T7 promoter-specific base pairs. Promoter activities of 28 sequences were measured in vitro with T7 an d SP6 polymerases separately under optimal conditions at 6 mM MgCl2. S ingle and double substitutions at -12 and -10 do not significantly aff ect the T7 promoter activity, although they are almost exclusively con served among T7 genomic promoters. Changes at -10 of SP6 promoter hard ly affect the activity. However, any T7 variants that contain either o r both changes at -9 and -8 show greatly reduced activity. Interesting ly, the double substitution at -9 and -8 yields significant SP6 promot er activities and virtually no T7 promoter activity. Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 p romoter activities, although they still show some SP6 promoter activit ies. However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity. N o other SP6 variants show any T7 promoter activity. All these results indicate that the 2 base pairs at -9 and -8 of both the T7 and SP6 pro moters are the primary (if not the only) determinants of Specificity a nd that the hierarchy of importance of positions for promoter activity is -8, -9 > > -10 > -12. Also, a phyLogenic relationship among the T3 , T7, K11, and SP6 promoters is suggested based on dissimilarities in their sequences from -12 to -8.