Constitutive function of a positively regulated promoter reveals new sequences essential for activity

Abstract

A consensus "-10" recognition sequence for RNA polymerase was created at the positively regulated lambda Pre promoter by introducing three single base pair mutations. This altered promoter, Pre*, functions constitutively in vivo and in vitro at high efficiency despite very poor consensus "-35" region sequence homology. We examined the influence of the -35 region sequence information on promoter function by shifting the wild type -35 region +/- 2 base pairs relative to the -10 region consensus sequence and by completely replacing it with alternative DNA sequences. In every case, the altered Pre* promoters retained transcriptional activity although differences in their transcriptional efficiencies were observed. Apparently the Pre* promoter does not require specific -35 region sequences for constitutive promoter activity, although the -35 region sequences can modulate overall promoter strength. In addition, by point mutation analysis we have identified bases immediately upstream of the -10 hexamer which are essential for constitutive function of the Pre* promoter. We propose that these mutants define an extended -10 region at Pre* that compensates for its poor -35 region sequence information by providing critical contacts that stabilize productive RNA polymerase binding.