Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters.

S.S Lee,C Kang

doi:10.1016/s0021-9258(19)36513-5

Abstract

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.

Highlights

From the Department o.f Li.fe Science,Korea Advanced Institute of Science and Technology, 373-1 Kusung-dong, Yusung-gu, Taejon 305-701, Korea
Bacteriophage T7 and SP6 RNA polymerases and RNA polymerase shares a highly conserved 20-23-base-pair their promoters share a high degree of their primary consensus sequence [5, 9,10,11,12]
As an initial study of the molecular basis for specific interactions between phage RNA polymerases and promoters, the T7 and SP6 systems were chosen in this study, because very variants that contain eitheorr both changesat -9 and little cross-reaction occurs between the two

Summary

TCGATTTAGGDSWCACTATAGAAG AAATCCHSWGTGATATCTTCCTAG

S and W indicate, respectively, 1:l mixtures of C and G and A and T. The annealed duplexes of T7 promoters containing 2-base wobbles in four positions were cloned into theAccIIBamHI site of pUC119. The prototype and 15 variants were selected and identified by sequencing about 100 base pairs around the promoter region in about 80 clones. I n Vitro Transcription-Transcription initiation efficiencies of phage promoter variants were measured by the in vitro transcription activity of intact plasmids [16]. Tris-HC1 (pH 8.0 for T7, and pH 7.5 for SP6), 6 mM MgClz, 2 mM Based on the above considerations, we chose base pairs at spermidine, 10 mM dithiothreitol, 0.3 mM each rNTP, 0.2 unit/pl -12, -10, -9, and -8 as possible candidates for the basis of RNase inhibitor RNasin (Promega), 20 nM circular 0.25 unit/pl phage RNA polymerase (Promega), and plasmid DNA, 0.3 p~ [(Y-~’P]. After specific base pairs in the four positions 30-min reactions, transcripts were precipitated by addition of 100 p1 of ice-cold 10% trichloroacetic acid. The transcription activity of each of the variants was determined in vitro using T7 RNA polymerase under the usual optimal conditions with 6 mM MgC12 and compared with the activity of the consensus sequence (Fig. 1)

RESULTS

DISCUSSION

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