Ca. Christoffersen et al., Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli, J BACT, 183(6), 2001, pp. 2059-2070
The overlapping and opposing promoter elements for the Escherichia coli fep
DGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protei
n) within the enterobactin gene cluster were investigated by measuring the
effects of site-specific mutations on transcript levels and on expression o
f reporter genes in a bidirectional transcriptional fusion vector, Primary
promoter structures for the opposing transcripts overlapped extensively suc
h that their -10 sequences were almost directly opposed on the two strands
of the DNA helix and their fl transcription start sites were only 23 bp apa
rt. Relative to the E, call consensus sequence, both promoters were poorly
conserved at the -35 position and mutations which strengthened the -35 elem
ent of either promoter significantly enhanced its transcription, decreased
that of the opposing promoter, and dramatically altered iron-mediated regul
ation of expression. Both the fepD and ybdA primary promoters were shown to
require a 5'-TGn-3' upstream extension of their -10 elements for optimal a
ctivities. Secondary promoters were identified for both fepD and ybdA, and
their contributions to the overall expression levels were evaluated in thes
e dual expression vector constructs. The data provided strong evidence that
the architecture of the regulatory elements within the overlapping fepD an
d ybdA promoters is configured such that there is a direct competition for
binding RNA polymerase and that the expression levels at these promoters ar
e influenced not only by the activity of the opposing promoters but also by
additional promoter sequence elements and perhaps accessory regulatory fac
tors. Iron-mediated regulation of these promoters through the repressor pro
tein Fur is a consequence of the relative promoter strengths and the positi
on of an operator site that consists of two overlapping Fur-binding sequenc
es in this compact regulatory region.