Upstream sequence activation of Escherichia coli argT promoter in vivo and in vitro

Abstract

Escherichia coli argT promoter in a galK fusion construct is shown by BAL 31 deletion to require its upstream region for high in vivo activity. The extent of activation conferred by the upstream sequence from -130 to -38 is 25-fold. A spontaneous mutant containing a T to G transversion at -37 (i.e., the T-37G promoter) shows a similar requirement; however, the upstream sequence producing a 10-fold effect spans only -130 to -60. The difference in upstream sequence boundaries between the wild-type and T-37G promoters suggests the possible existence of two activating elements. Gel mobility investigation points to the presence of bent DNA in the argT promoter, and the bent center was localized to the -90 to -95 region by circular permutation analysis. The role of the upstream activating sequence (UAS) in promoter function was probed by competitive transcription experiments in vitro. Results of this type of analysis indicate that the full UAS activates transcription through a combined effect on KB and k2. Of these, KB is significantly strengthened by the proximal element, and k2 is stimulated to a smaller extent by the distal element. The evidence from deletion analysis, gel mobility investigation, and competitive transcription together support a "two-element" model of UAS function for the argT promoter.