Two 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 high degree of their primary structure homology, but each polymerase exclusively recognizes its own promoter sequence. To reveal the molecular basis of this specificity, 4 base pairs at positions -12, -10, -9, and -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 and SP6 polymerases separately under optimal conditions at 6 mM MgCl2. Single and double substitutions at -12 and -10 do not significantly affect the T7 promoter activity, although they are almost exclusively conserved among T7 genomic promoters. Changes at -10 of SP6 promoter hardly affect the activity. However, any T7 variants that contain either or both changes at -9 and -8 show greatly reduced activity. Interestingly, the double substitution at -9 and -8 yields significant SP6 promoter activities and virtually no T7 promoter activity. Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 promoter activities, although they still show some SP6 promoter activities. However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity. No 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 promoters are the primary (if not the only) determinants of specificity and 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.
Lee
S S
SS
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
Kang
C
C
D017123:Bacteriophage T7 / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases