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J. Biol. Chem., Vol. 277, Issue 3, 2169-2175, January 18, 2002
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From the The occupation of the
In Vivo UV Laser Footprinting of the
Pseudomonas putida
54 Pu
Promoter Reveals That Integration Host Factor Couples
Transcriptional Activity to Growth Phase*
,¶
Department of Microbial Biotechnology,
Centro Nacional de Biotecnología, Consejo Superior de
Investigaciones Científicas, Campus de Cantoblanco,
28049 Madrid, Spain and § Enzymologie et Cinétique
Structurale, UMR 8532 du CNRS, Institut Gustave Roussy, 94805 Villejuif, France
54-dependent Pu promoter
of Pseudomonas putida by the integration host factor (IHF)
under different growth conditions has been monitored in its native
state and stoichiometry (i.e. monocopy) with UV laser
footprinting technology. We present evidence that an abrupt change in
intracellular IHF concentrations occurs when P. putida
cells enter stationary phase. This change results in enhanced binding
of the factor to the promoter and in the ensuing bending of the target
DNA. Since Pu activity depends rigorously on DNA bending,
promoter occupation is in turn translated into a much higher
transcriptional output when cells leave exponential growth. Inspection
of the residual activity of Pu in an
IHF
strain reveals that IHF predominantly locks
the capacity of the promoter to specific growth stages and also that
additional physiological signals are entered in the system through
54-RNA polymerase. The results substantiate the
notion that 54 promoters process metabolic co-regulation
signals through factor-induced changes in the architecture of the
cognate DNA region. Further, they validate UV laser technology as a
suitable tool to visualize nondisruptive alterations of DNA shape
in vivo.
*
This work was supported by European Union Contracts
QLK3-CT2000-00170 and QLK3-CT1999-00041, by Grant BIO98-0808 of the
Spanish Comisión Interministerial de Ciencia y Tecnología
(CICYT), and by the Strategic Research Groups Program of the Autonomous
Community of Madrid. The support of the Fondation de la Recherche
Medicale (FRM) for the award of a grant (to M. B.) for the
establishment of a new laboratory is acknowledged.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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