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J Biol Chem, Vol. 274, Issue 47, 33790-33794, November 19, 1999

Recruitment of RNA Polymerase Is a Rate-limiting Step for the Activation of the ⁵⁴ Promoter Pu of Pseudomonas putida

Manuel Carmona, Víctor de Lorenzo, and Giovanni Bertoni^¶

From the  Department of Microbial Biotechnology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain and ^¶ Dipartimento di Genetica e Biologia dei Microrganismi, Universitá degli Studi di Milano, via Celoria 26, 20133 Milan, Italy

The activity of the 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 XylR only from solution. Although the transcription activation pathway in this system lacked the step of integration host factor (IHF)-mediated looping of the XylR·DNA complex toward the prebound RNA polymerase, IHF still stimulated promoter activity. The positive effect of IHF became evident not only with XylR from solution, but also with other 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 XylR-mediated formation of an open complex.

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