Transcript j17Chapt_17_bactGene..

Lecture 20
Control of gene expression in bacteria.
Chapter 17
RNA polymerase
The CAP protein recruits RNA polymerase
to the Lac operon promoter
1
J G Burr, 10/27/13
The amount or activity of any protein found inside a
bacterial cell could be governed in any of 3 ways:
(1) By regulating the ability of RNA polymerase to transcribe
mRNA for that protein (transcriptional control)
(2) By regulating the ability of ribosomes to translate the mRNA
into protein (translational control)
(3) By regulating either the lifetime of the protein in the cell, or
its specific biological (enzymatic) activity (post-translational
control)
2
J G Burr, 10/27/13
We are going to focus on transcriptional control.
And in particular, we are going to return to a
discussion of the Lac operon, which we first discussed
in the context of the discovery of the promoter.
3
J G Burr, 10/27/13
You may remember we said earlier that when E. coli is put
in growth medium containing both glucose and the
disaccharide lactose, it will use up all the glucose before
starting to metabolize the lactose.
Fig 14.2 from 1st Ed; not in current Ed.
Diauxic growth
4
J G Burr, 10/27/13
And we learned that Jacob and Monod found that, after all the
glucose had been used up, the presence of lactose causes the
expression of two genes, lacZ (-galactosidase) and lac Y
(galactosidase permease). The tandem pair of lacZ and lac Y genes,
coordinately expressed, was called the “lac operon”. They also
discovered a third gene that regulated the expression of lacZ and
lac Y. They named this regulatory gene lac I.
Galactoside
permease
Equiv to Figs 17.4 + 17.5 in 3rd Ed.
LacY product
5
J G Burr, 10/27/13
And we learned that they then discovered that two other genetic
elements are present immediately in front of the lacZ gene, the
“promoter” and the “operator”. The promoter was defined as the
location where RNA polymerase sits down to begin transcribing the
lac operon.
RNA Polymerase binds to
the promoter to initiate
transcription of mRNA
Fig 14.8, 1st Edition of your text
6
J G Burr, 10/27/13
The operator was a sequence on the DNA, located between the
promoter and the lacZ gene, where a “repressor” protein (encoded
by the lacI gene) can bind. In the absence of lactose, the repressor
binds to the operator, and blocks the movement of RNA polymerase.
In absence of
the disaccharide
lactose:
No transcription
of the genes
needed to
metabolize
lactose.
Lactose binds to the repressor, thereby inactivating it, and
permitting RNA polymerase to transcribe the Lac operon.
When lactose
is present:
Fig 14.7, 1st Edition of your text
Transcription of
the genes needed
to metabolize
lactose!
7
J G Burr, 10/27/13
Consider now what the situation would be in cells with a
mutant lacI gene (lacI -) that cannot produce a functional
lac repressor protein:
In this case,
the
transcription
of the lac
operon is
constitutive;
ie,
transcription
is continuous
and ongoing
regardless of
whether or
not lactose is
in the growth
medium.
8
J G Burr, 10/27/13
Well, all this explains why, under normal circumstances, the genes for
making lactose-metabolizing enzymes are not expressed unless
lactose is actually present in the growth medium.
No lactose
Lactose
present
But it doesn’t explain why the cell
doesn’t turn on expression of lac
operon if glucose is present at the
same time as lactose; ie, why does
the cell wait until all the glucose
is used up before permitting lac
operon expression?
Growth on lactose
only after glucose
is all used up.
9
J G Burr, 10/27/13
More explicitly:
Here, they’re
growing on lactose.
Here, cells are
using only glucose
to grow.
Here, lactose is present, but the lac
operon is shut down. Why? Lactose
would be binding the repressor and
inactivating it. So what is keeping
the RNA polymerase from
transcribing the LacZ and LacY
genes?
This part makes sense, because here
the lac operon is expressed.
Lactose is binding the repressor and
inactivating it. Therefore RNA
polymerase is transcribing the LacZ
and LacY genes, and the cell is able
to grow with lactose as a carbon
source.
10
J G Burr, 10/27/13
This is where the CAP protein comes into the story. But before we
talk about CAP, you need to know about cyclic AMP (cAMP)
Cyclic AMP is a signaling
molecule used by both
prokaryotic and eukaryotic
cells. In response to events
in the external environment,
an enzyme called “adenylyl
cyclase” is activated, and it
converts ATP to cyclic AMP
(cAMP)
(cAMP has been called “an ancient
starvation signal” in bacteria)
Cyclic AMP
11
J G Burr, 10/27/13
There is another enzyme, called a “phosphodiesterase” that degrades
cAMP to plain old AMP.
So the level of cAMP in a cell at any
moment is governed by the relative
activities of the adenylyl cyclase
that makes cAMP, versus the
phosphodiesterase that destroys
cAMP
If adenylyl cyclase is relatively
inactive, then the ongoing activity
of the phosphodiesterase will
destroy any cAMP in the cell; if
adenylyl cyclase becomes
activated (by phosphorylation, for
example) then the production of
cAMP will outstrip the ongoing
slow degradation of it by the
phosphodiesterase.
12
J G Burr, 10/27/13
Now then, as we have said, E coli (and other bacteria) keep the lac
operon shut down as long as glucose is present. It was thought that a
breakdown product of glucose metabolism (a “catabolite” of glucose)
might be involved in the repression, and so this repression of the lac
operon (and many other operons for alternate energy sources) was
called “catabolite repression”
It turns out that catabolite
repression (the presence of
glucose) is instead mediated by
the absence of cyclic AMP.
(In the absence of glucose, a
bacterial “phosphotransferase”
protein [“IIAGlc”] accumulates in its
activated form, and begins to
phosphorylate (and thereby activate)
Adenylyl Cyclase. Adenylyl cyclase
therefore begins synthesizing cAMP
at a high rate. Levels of cAMP rise in
the cell.)
13
J G Burr, 10/27/13
To summarize:
Glucose present: no cAMP; repression of lac operon
(“catabolite repression”)
Glucose present in growth medium
Glucose absent: high levels of cAMP; lac operon can
be expressed, if lactose is present
No Glucose in medium
cAMP
14
J G Burr, 10/27/13
In the 1960’s it was discovered that cAMP acts by binding to a
protein called the “catabolite activator protein” CAP. And in fact, it
became clear that the process which had been named “catabolite
repression” in fact involves positive control of the lac operon,
mediated by CAP, which occurs in the absence of glucose.
When cAMP binds to it, CAP is in turn able to bind DNA at a
specific site upstream of the promoter, called the “CAP site”.
When CAP binds the DNA, it induces a bend in the DNA; this
bend in the DNA distorts the double helix, and greatly increases
the ability of RNA polymerase to sit down on certain promoters.
CAP also interacts with RNA
polymerase, and stabilizes
its (otherwise weak) binding
to certain promoters (like
the lac promoter).
cAMP
cAMP
cAMP
cAMP
Green = the CAP protein
15
J G Burr, 10/27/13
Sidebar: There is plenty of CAP protein around in the cell at all times,
but if the cell is not starving for glucose, all this CAP protein is
inactive (ie, unable to bind to DNA); as we have been saying, it is only
when there is no glucose available that the cell makes cAMP, and this
cAMP then binds to pre-existing CAP, thereby activating CAP.
So CAP protein activity is not regulated at the transcriptional
or translational level; ie, the cell is constantly making plenty of
mRNA for CAP, and translating CAP mRNA into CAP protein.
Rather, this is an example of a protein whose activity (we would
say) is “regulated at the post-translational level.”
cAMP
cAMP
cAMP
cAMP
Green = the CAP protein
16
J G Burr, 10/27/13
Here are two representations of the actual structure of
CAP (with bound cAMP) binding to DNA and bending it.
Side chains on an alpha-helical segment interact
with specific bases in the DNA
cAMP
The CAP protein is a dimer; each subunit binds 2 molecules of
cAMP. It is a member of a class of DNA binding proteins
called “helix-turn-helix” proteins.
17
J G Burr, 10/27/13
Sidebar: the “helix-turn-helix” motif is found in many
DNA-binding proteins. Here is more detail:
Side chains on an alpha-helical segment (the
“recognition helix”) interact with specific bases in
the DNA
“Major groove”
“Minor groove”
18
J G Burr, 10/27/13
Here are some other examples of helix-turnhelix DNA-binding proteins:
(Note that many of these helix-turn-helix DNA-binding
proteins function as dimers.)
(For the test, just know that “many proteins that
bind DNA do so via a ‘helix-turn-helix’ domain, and
they do so by binding in the major groove of the
DNA double helix.”)
19
J G Burr, 10/27/13
The lac repressor also binds to the operator DNA via a
helix-turn-helix motif. It turns out, by the way, that
the lac repressor functions as a tetramer:
Tetrameric lac
repressor
20
J G Burr, 10/27/13
It further turns out that there are two other
operator sequences, O2 and O3, flanking the
originally identified operator (O1). Since helix-
turn-helix proteins (and other DNA-binding proteins)
generally interact with nucleotide sequences having
‘dyad symmetry’, it comes as no surprise that all three
of these lac operator sequences have dyad symmetry.
21
J G Burr, 10/27/13
The tetrameric
repressor binds
simultaneously to
either O1 plus O2,
or O1 plus O3,
thereby producing
a loop in the DNA:
(Repressor
tetramer
comes off
of DNA
when
inducer
molecules
bind it
22
J G Burr, 10/27/13
Returning again, now, to the relationship
between glucose, cAMP, and CAP, here is a
summary figure :
(This figure is taken from the
2nd edition of your book)
23
J G Burr, 10/27/13
More detail on the role of CAP in gene expression:
The expression of genes like the lac operon is almost completely
dependent on the binding of CAP. CAP is said to mediate “positive”
gene regulation.
No glucose =
high levels of cAMP=
CAP active
Glucose present =
low levels of cAMP=
CAP inactive
24
J G Burr, 10/27/13
So the expression of the lac operon is under both
positive (CAP-mediated) and negative (lac repressor)
control.
In order for the lac operon to be expressed, one must have both
the presence of active CAP and the absence of active lac repressor
Presence of lactose
in the medium
Absence of glucose in
the medium ( cAMP)
The CAP protein binds RNA
polymerase, and stabilizes the binding
of RNA polymerase to the promoter.
(See slide #1 of this lecture.)
25
J G Burr, 10/27/13
So all this time when we’ve been showing this picture
(for example, back on slide #6),
the implication has been that if the Repressor isn’t bound to the Operator,
RNA polymerase will bind to the promoter, and Lac operon will be transcribed.
But in fact, the Lac operon promoter is a weak promoter (poor consensus
sequence) (RNA polymerase binds with low affinity), and in the absence of the
CAP protein, just low, basal levels of transcription occur.
One must have both bound CAP protein and the absence of active lac
repressor to get high level expression of the Lac operon!
26
J G Burr, 10/27/13
The CAP protein recruits RNA polymerase to the
Lac operon promoter:
RNA polymerase
cAMP
cAMP
cAMP
cAMP
Not only does CAP induce a strong bend in the DNA
(shown earlier [fig 14], but not here), but also by
physically binding both the DNA and the RNA
polymerase, it acts to stabilize the interaction of the
polymerase with the promoter.
27
J G Burr, 10/27/13
Summary:
(Because glucose is present, cAMP is not made, and therefore CAP is inactive)
If there’s glucose, but no
lactose, cell won’t make
enzymes for lactose
metabolism
If there’s glucose, and
also lactose, cell won’t
make hardly any enzymes
for lactose metabolism;
instead, it will use up all
the glucose first
If there’s no glucose
(signaled by high cAMP),
but there is lactose, cell
will make enzymes for
lactose metabolism
If there’s no glucose
(signaled by high cAMP),
but also no lactose, cell
won’t make enzymes for
lactose metabolism
28
J G Burr, 10/27/13
Here’s another summary, taken from another book:
cAMP
cAMP
Alberts, fig 7-38
No glucose
means high
levels of
cAMP = CAP
binding
29
J G Burr, 10/27/13
And that is the
molecular explanation
for the diauxic growth
that E. coli exhibits
when placed in a medium
containing both glucose
and lactose.
30
J G Burr, 10/27/13
Jacob, Monod, and André Lwoff won the 1965 Nobel
Prize for their contribution to working out this
story.
The Nobel Prize in Physiology or Medicine 1965
"for their discoveries concerning genetic control of enzyme and virus synthesis"
François Jacob
(For the test, know the
names Jacob and Monod as
the “Lac operon guys.’
Jaques Monod
André Lwoff
For personal anecdotes about these guys, go to
http://www.dnaftb.org/dnaftb/33/concept/
31
J G Burr, 10/27/13