Download the lac and tryp operons in E. coli

Please Come to your
Assigned Exam Room!
A-F Salomon 101
G-L Biomed Center 202
M-Z Salomon 101
Corrections to
Monday’s Lecture
(my 3’ and 5’ mixup)
Lecture Capture is
now working!
I’ve posted corrected
slides (and a narrated
video) to atone for my 3’
and 5’ mixup on Monday.
Exam 2: Friday, March 15th.
Exam 2 (3/15) will include a metabolic
pathways handout
Handout Copy is
on Web Site
Control of Gene Expression
Model Systems
The Lac Operon
The Tryp Operon
Positive & Negative Control
Eukaryotic Gene Regulation
1) How does a single gene
produce a trait which we
can see or measure? Such
as blood type or flower
color or seed shape?
2) If all cells contain the
same set of genes, how do
the genes in different cells
of an organism produce so
many different cell types?
Is the expression of genes
controlled?
Needed: A Model System for the
mechanism by which gene expression
is controlled
(the lac and tryp operons in E. coli )
Escherichia coli
Human intestinal bacterium.
4 million base pairs of DNA.
4,000 genes.
Widely used as a
model system for:
1) Genes & Traits
2) Control of gene
expression.
One of E. coli’s most easily
observed traits is the ability
to grow on certain food
sources. For example, the
disaccharide lactose.
In order to grow on lactose, the cell must produce a
protein enzyme known as -galactosidase.
But it makes no sense for the
bacterium to produce this
enzyme if it is not in an
environment where lactose is a
common food source.
Lactose is, in fact,
“inducible.” Significant
amounts of the enzyme
are made only when
lactose is added to the
growth medium.
The lac operon
Investigated by Jacob and Monod in
Paris. Three genes “operated”
together = an “operon.” The first
system in which regulation of gene
expression was worked out (all by
classic genetic and biochemical
techniques in the 1960s).
ONPG (Synthetic Inducer)
o-nitrophenyl--D-galactoside)
Organization of the lac Operon
3 proteincoding
genes
P
O
Promoter
Operator
Z
Z = b-galactosidase
Y = lac permease
A = transacetylase
Y
2 control
regions
A
lac Operon:
RNA polymerase binds
to the Promoter DNA
sequence. It them
moves past the
Operator region, and
transcribes the 3 genes
into a single mRNA
molecule.
P
O
Z
AUG
The lac mRNA molecule
contains 3 sets of start
and stop signals for
translation, producing 3
different protein
products.
start
UAG
stop
A
Y
AUG
UAG
AUG
UAG
P
O
Z
A
Y
Z = -galactosidase
Y = permease (helps the lactose to enter cell)
A = transacetylase (puts acetyl groups on sugars)
lac
Y
Y
Y
Y
glu
lac
Z
gal
P
O
i
Z
Y
A
Binding of the lac repressor protein
(i) prevents RNA polymerase from
moving past the operator to reach
the structural genes.
P
O
Z
Y
i
lac
i lac
A
Lactose acts as an
“inducer,” binding to the
repressor and causing a
conformational change —
making the repressor
unable to bind to DNA.
The lac repressor system responds automatically to
the presence of lactose.
Lactose absent? No transcription (saving the cost of
making proteins that are not needed)
Lactose present? The operon is transcribed
(enabling the cell to use lactose as an energy source).
The lac repressor is
a DNA-binding
protein that
recognizes the base
sequence of the
Operator region.
Another example of gene
regulation is the tryp
operon.
Tryptophan is an amino
acid produced in a pathway
that involves 5 enzymes.
These 5 enzymes are
coded for by five genes
found in the tryp operon.
The tryp repressor is coded by the trpR
gene. However, the repressor is inactive by
itself (unable to bind to DNA)
The tryp repressor must first bind to tryptophan itself
(serving as a corepressor) before it can bind to
operator DNA to block transcription of the operon.
P
O
Z
A
Y
Lac is an inducible operon
P
O
E
D
CY
B
A A
Tryp is an repressible operon
Tryp and lac are both examples of
negative control (since all the repressor
can do is to block transcription.)
A positive control mechanism would
enhance transcription.
From your
Textbook:
• The distinction
between Positive
and Negative
control is whether
the DNA-binding
protein blocks or
triggers
transcription
glu
lac
Y
Y
Y
Y
glu
lac
glu
Z
gal
The “cost” of using lactose as a food source is
considerable — it requires the synthesis of Z and Y
proteins. Glucose provides just as much energy,
and doesn’t require either protein. Therefore it
makes sense for the cell to “prefer” glucose to
lactose.
.The
. . so,
coli hasalso
a system
that “senses”
the
lacE.operon
provides
an example
level of glucose. (smart bug!)
of positive control
The system is built around a region near the promoter to which a
protein known as CAP (catabolite-activated protein) can bind.
CAP bends the DNA in the Promoter region, dramatically
increasing its affinity for RNA polymerase.
CAP protein bound
to DNA
What I’ve neglected to mention (intentionally) is that the
lac Promoter actually has only a very weak affinity for
RNA polymerase. So, even when it is induced by lactose,
there’s only a low (basal) level of transcription.
In the presence of glucose, the CAP protein does not
bind and the operon is not fully activated.
The CAP protein is activated by a small molecule called
cyclic AMP (cAMP) . . . . cAMP levels rise when glucose is
scarce (and fall when it is abundant).
Rising cAMP levels (when glucose or other food molecules
are scarce) result in Frequent Transcription.
Low cAMP levels? Much less frequent transcription.
This means that efficient transcription of
the operon only occurs when glucose is
low (causing cAMP to be high), causing
CAP to bind; and when lactose is present
(causing the repressor to release from the
Operator)
Termination of Transcription takes place by at least
4 different mechanisms. Transcription in the Tryp
operon is terminated by the formation of a loop in
mRNA that triggers RNA polymerase to release the
new mRNA molecule.
Gene Expression in Eukaryotes
Similarities to Prokaryotes:
1) Promoters (RNA polymerase binding sites)
2) Repressors (by many different names)
3) Enhancers (by many different names)
Complications:
1) Lots more DNA (human: 1000x as much as E. coli)
2) DNA extensively bound to proteins (histones & nonhistones)
3) Multiple levels regulating gene expression the rule
rather than the exception
4) 3 forms of RNA Polymerase (I, II, III) (II = mRNAs)
The start of
Transcription in
Eukaryotes involves
the binding of
multiple factors.
To form a
Transcription
Complex.
Eukaryotic mRNAs:
• Are processed by intron removal.
• Have a 5’ cap attached.
• Have a 3’ poly-A tail.
Gene Expression in
Eukaryotes can be
regulated at multiple levels
(including the organization
of DNA & protein in the
nucleus, transcription, RNA
processing, exit from the
nucleus, translation, and
protein turnover in the
cytoplasm) . . . .
. . . . and, by a number of
newly-discovered
mechanisms, including RNA
interference (RNAi)
(discovered by Nobel
Laureate Craig Mello ’82.