Recruitment of RNA polymerase is a rate-limiting step for the activation of the σ54 promoter Pu of Pseudomonas putida

The activity of the sigma(54)-promoter Pu of Pseudomonas putida was examined in vitro with a DNA template lacking upstream activating sequences, such that RNA polymerase can be activated by the enhancer-binding protein Xy1R only from solution. Although the transcription activation pathway in this system lacked the step of integration host factor (IHF)-mediated looping of the Xy1R DNA complex toward the prebound RNA polymerase, IHF still stimulated promoter activity. The positive effect of IHF became evident not only with Xy1R from solution, but also with other sigma(54)-dependent activators such as NtrC and NifA Furthermore, an equivalent outcome was shown for the nonspecific DNA-binding protein HU. This stimulation of transcription in the absence of the enhancer was traced to the recruitment of RNA polymerase (i.e. increased efficiency of formation of closed complexes) brought about by IHF or HU binding. Thus, under limiting concentrations of the polymerase, the factor-mediated binding of the enzyme to Pu seems to enter a kinetic checkpoint in the system that prevents the Xy1R-mediated formation of an open complex.