Effects of multiple mutations at the conserved TATA sequence of bacteriophage SP6 promoter on transcription efficiency.

Mutations of A<==>T were introduced individually and multiple to TATA from -4 to -1 of the phage SP6 promoter and their effects on transcription initiation efficiency measured in vitro. All 15 mutants tested were less active than the wild type. Mutation at -4T nearly abolishes promoter activity independent of other changes, and alteration at -3A reduces promoter activity substantially. On the other hand, effects of mutations at -2T and -1A depend on other changes, suggesting their role should be associated with neighboring base pairs. These results suggest that -4T and -3A are involved in SP6 RNA polymerase binding and -2T and -1A are involved in DNA unwinding. This bipartite role of the SP6 promoter TATA contrasts with the single role of T7 promoter TATA on DNA unwinding. The polymerase binding region extends further downstream in the SP6 promoter than in the T7 promoter.