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repressor and derepression of the constituent genes of the LexA regulon.
Ronen
et al.
(2002)
fused the promoter regions of eight LexA regulon genes (
uvrA
,
uvrD
,
lexA
,
recA
,
ruvA
,
polB
,
umuD
and
uvrY
) to a promoterless
gfpmut3
gene and established
the fusion constructs in
E. coli
on a low copy number (pSC101-based replicon) plas-
mid. Several important features of the dynamics of SOS response induction emerged
from this study. The activities of all eight promoters increased within minutes of UV
light exposure, each with a distinctive kinetic profile and expression level. The
kinetic profiles were consistent with a less-temporally refined transcriptomic anal-
ysis, supporting the accuracy of this reporter fusion-based study (
Courcelle
et al.
,
2001
). Promoter activities increased for approximately 20 min and then decreased
with distinctive kinetics. The order in which the genes were turned off reflected their
function in SOS-mediated DNA repair: the expression of genes encoding nucleotide
excision repair functions (
uvrA
) and the RecA and LexA regulators (
recA lexA
) was
turned off first, while genes (
polB
,
uvrD
and
ruvA
) encoding proteins involved in
other repair processes of the SOS response were turned off later. Thus a temporal
correlation was shown to exist between the kinetics of promoter activity and the
order of events during DNA repair and the restoration of DNA replication. The
authors also showed a close correlation between the level of active LexA protein
(determined experimentally by Western blotting analysis) and that inferred from
the activities of the constituent regulon promoters. Thus promoter activity is a good
indicator of the activity of transcriptional regulators
in vivo
.
A subsequent study from the Alon group established promoter activities for
operons that encode the enzymes for several amino acid biosynthetic pathways.
The rationale for this study is that each amino acid biosynthetic pathway constitutes
an individual regulatory network. Thus by determining promoter activities for
operons that encode the enzymes of each amino acid biosynthetic pathway at high
temporal resolution, it is possible to establish regulatory principles for how these bio-
synthetic pathways are controlled and how they are networked. The activities of 52
promoter fusions were established at high temporal resolution in this study.
Promoter-containing fragments were cloned into a low copy number (pSC101-based
replicon) plasmid containing promoterless
gfpmut2
(pUA66) or
luxCDABE
(pUAL94) genes. Several novel features of the regulation of amino acid biosynthesis
were revealed. As expected, promoters are generally turned off when all amino acids
are present in the medium and turned on when specific amino acids are absent. Addi-
tion of one amino acid generally leads to repression of the promoters of the operons
that encode its synthesis. However, cross-regulation was also observed: in some
cases, addition of a particular amino acid leads to altered expression of operons that
encode the synthetic enzymes of other amino acids. Thus promoter fusion analysis
reveals the existence of a complex web of cross-regulation of amino acid biosynthe-
sis in
E. coli
. Such analyses also reveal the temporal sequence of promoter activation
that occurs when a particular amino acid is removed from the medium. Promoters are
activated in a sequence that sees those for genes encoding enzymes acting early in the
pathway being turned on first, while those for genes encoding enzymes acting later in
the pathway are turned on up to 10 min later (
Zaslaver
et al.
, 2004
).
