Download hypothesize that AraC can exist in 2 states, P1 and P2

-what’s going on?
-hypothesize that AraC can exist in 2 states, P1 and P2
-P1 – in absence of arabinose; functions as a repressor
-P2 – in presence of arabinose; functions as an activator
-AraCc mutant is locked in P2 form, but in a merodiploid F’
araC+/araCc, wildtype AraC represses expression in absence of
arabinose, preventing activation by AraCc
Identification of an Operator for the ara operon
-deletions that extended beyond araC caused increased basal
expression of the araBAD operon
-propose that deletion removes an operator
-now know that AraC binds to three sites in the araBAD regulatory
region, araI, araO1, and araO2
-in absence of arabinose, AraC binds araO2 and araI and represses
araBAD transcription
-arabinose alters AraC such that repression is alleviated and
transcription is initiated (requires a second activator, CAP)
-in addition, AraC binds to araO1 in absence of arabinose to
repress its own transcription
CAP/CRP
-Catabolite Activator Protein (CAP) or Catabolite Repression
Protein (CRP)
-transcription activator that controls expression of genes involved
in carbon and energy source utilization
-since glucose is the preferred carbon source of E. coli, CAP
ensures that other carbon utilization pathways are not expressed in
the presence of glucose CATABOLITE REPRESSION
-accordingly, CAP functions as a transcription activator of genes
involved in metabolism of alternative carbon sources when glucose
is NOT present (ie. lac, ara, gal, mal operons)
-it does this by sensing the levels of cAMP (cyclic AMP)
-when glucose is being metabolized, levels of cAMP (cyclic AMP)
are decreased
-conversely, when glucose is not being metabolized levels of
cAMP are elevated
-CAP is a symmetrical dimer of two identical subunits
-when bound to cAMP (low glucose, high cAMP), CAP is active
and binds to a specific palindrome found upstream of genes that
are controlled by catabolite repression
-consensus: 5’-AAATGTGATCT-AGATCACATTT-3’
-DNA binding mediated by a HTH present in each subunit and
leads to DNA bending (crystal structure)
Different CAP promoters
-CAP-dependent promoters can be grouped into 3 classes based on
location of the CAP binding site
i)
Class I
-require only CAP for transcription activation
-CAP binding site is upstream of RNAP binding site
-binding site can be located at various distances from start site as
long as it is on the same face of the helix as the RNAP binding site
(-61.5, -72.5, -82.5, -92.5)
-protoype is lacP1 promoter
ii) Class II
-require only CAP for transcription activation
-CAP binding site overlaps RNAP binding site
-replaces –35 binding determinants for RNAP
-prototype is galP1 promoter (CAP binding site at –41.5)
iii) Class III
-require a regulon-specific activator in addition to CAP for
transcription activation
-CAP binding site usually located more than 90 bp from
transcription start site
-eg. araBAD promoter
-much work suggests that the mechanisms of transcription
activation vary at Class I, Class II, and Class III promoters