Activation of the lac promoter and its variants. Synergistic effects of catabolite activator protein and supercoiling in vitro

Abstract

Escherichia coli lac promoter variants are shown to be subject to large synergistic transcriptional activation by catabolite activator protein (CRP) and DNA supercoiling in vitro. Activation was studied for the lac wild-type promoter, a promoter with a variant spacing (lac delta l) and two promoters with variant -10 regions (lac ps, lac UV5). The variant promoters respond to the simultaneous presence of CRP and supercoiling by exhibiting large multiplicative activation at the low to moderate superhelicities that are most pertinent in vivo. Although all four promoters can be activated by CRP, those made stronger by changing downstream promoter elements are less CRP-activated even though each contains an identical CRP binding site. When each of the variant promoters is made stronger by introducing DNA supercoils, the apparent CRP activation initially remains constant but eventually declines at higher superhelicities. Thus, strengthening the lac promoter through either DNA sequence changes or the introduction of high-level DNA supercoiling can lead to diminished potential for activation by CRP. These results are interpreted in terms of a role for CRP in providing extra stabilizing contacts for RNA polymerase binding that are necessary only when other stabilizing features of promoter structure are lacking.