Mm. Gonzalez-perez et al., Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants, J BIOL CHEM, 274(4), 1999, pp. 2286-2290
The Pm promoter, dependent on TOL plasmid XylS regulator, which is activate
d by benzoate effecters, drives transcription of the meta-cleavage pathway
for the metabolism of alkylbenzoates. This promoter is unique in that in vi
vo transcription is mediated by RNA-polymerase with different sigma factors
. In vivo footprinting analysis shows that XylS interacted with nucleotides
in the -40 to -70 region, In vivo and in vitro methylation of Pm shows ext
ensive methylation of T at position -42 in the bottom strand, suggesting th
at 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 mor
e 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 targe
t as two direct repeats with the sequence TGCAN(6)GGNCA. 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 t
he most critical for appropriate XylS-Pm interactions.