TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility

Abstract

The nmt1 gene of Schizosaccharomyces pombe is highly expressed and subject to transcriptional repression by thiamine. The nmt1 promoter, in common with other strong promoters in this organism, contains a canonical sequence element, 5'-ATATATAAA, located 25 bp upstream from the transcription start point (tsp). We have made stepwise deletions of the TATA box and quantitated the effects of the mutations by assaying the expression of the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) cloned downstream. Our results demonstrate that progressive truncation of the TATA box results in a concomitant decrease in promoter strength as judged both by the loss of CAT activity in cell extracts and by a reduction in the steady-state level of cat mRNA. Both the induced level and the residual, repressed level of expression observed in the presence of thiamine are similarly down-regulated. On the other hand, even in the most extreme mutant, the tsp is unaffected, suggesting that other elements in the nmt1 promoter are important in determination of the tsp. The properties of the modified promoters have made them useful for extending the range of the pREP inducible expression vectors.