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J Biol Chem, Vol. 274, Issue 4, 2286-2290, January 22, 1999
From the Department of Biochemistry and Molecular and Cellular
Biology of Plants, Estación Experimental del Zaidín,
Consejo Superior de Investigaciones Científicas, Apartado de
Correos 419, E-18008 Granada, Spain
The Pm promoter, dependent on TOL plasmid XylS
regulator, which is activated by benzoate effectors, drives
transcription of the meta-cleavage pathway for the
metabolism of alkylbenzoates. This promoter is unique in that in
vivo transcription is mediated by RNA-polymerase with different
sigma factors. In vivo footprinting analysis shows that
XylS interacted with nucleotides in the
Critical Nucleotides in the Upstream Region of the
XylS-dependent TOL meta-Cleavage Pathway Operon
Promoter as Deduced from Analysis of Mutants
40 to 70 region. In
vivo and in vitro methylation of Pm shows extensive methylation of T at position 42 in the bottom strand, suggesting that
it represents a key distortion point that may favor XylS/RNA polymerase
interactions. Methylation of T
42 was highest in cells
bearing XylS and in the presence of an effector. Gs in the 47 to 61
region appeared to be more protected in cells harboring XylS in the
presence than in the absence of the effector. Almost 100 mutants in the
Pm region between 41 and 78 were generated; transcriptional
analysis of these mutants defined the XylS target as two direct repeats
with the sequence TGCAN6GGNCA. These motifs cover the 70
to 56 and the 49 to 35 regions. Single point mutations revealed
that nucleotides located at 49 to 46 and at 59, 60, 62, and
70 are the most critical for appropriate XylS-Pm interactions.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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