Function of TATA sequence in bacteriophage promoters

Abstract

The role of TATA sequence from -4 to -1 of T7 and SP6 promoter in transcription efficiency was investigated with promoter variants carrying all the possible multiple and single mutation at this region having A:T or T:A base pair. Under supercoiled template conditions in vitro transcription activities of T7 mutant promoters were not reduced significantly compared to the wild type at 37℃ and 25℃. However when linear templates are used, the relative transcription activity of each mutant promoter varies substantially depending on its sequence. All mutants are less active than the wild type and generally mutants needing more free energy for melting are less active especially at lower temperature 25℃. It means that instability of this region affects transcription activity. Unexpectedly, activities of the mutants having the same free energy change but different sequences are diverse, which means that there is also a sequence effect on transcription activity besides melting energy effect of DNA duplex itself. Within the mutant promoters having similar DNA duplex stability, the mutations at -4 and -3 make promoter substantially more weak in promoter activity than the mutations at -2 and -1. In contrast to results of T7, transcription activities of SP6 promoters at supercoiled condition are diverse depend on its sequence. Alteration at -4 reduces promoter activity seriously and change at -3 also reduces the activity even though the effect is less than that of -4. Effect of base pair alterations at -2 and -1 are relatively less than those of -4 and -3. When linear templates are used, the relative transcription activities of all mutant promoters are diminished less than 20%. So, it can be suggested that TATA sequence in T7 promoter concerns major melting of DNA duplex and that of in SP6 promoter does not only melting but also specific binding with RNA polymerase.