AP Biology
Molecular Genetics
Regulation of Gene Expression in Prokaryotes
Chapters 18 – Section 18.1, pages 351-356
First, some review:
• One Gene-One Enzyme Hypothesis
• Beadle & Tatum identified genes as being responsible for the
production of enzymes
• Central Dogma of Molecular Genetics
The need for Regulation
• Cells have hundreds of genes, but will not need all of those
proteins all of the time.
• The enzymes a cell needs at any given time will be a function
of the environment and conditions it is exposed to
• In a multicellular organism, each cell has identical genetic
material but produces only the proteins it needs to function.
• Differentiation of cells into a variety of tissues results from
variations in gene activity
• For example, muscle cells will express the genes for actin and
myosin, but not for hemoglobin or insulin.
The Operon Hypothesis
• Sounds like a Jason Bourne movie, but actually a theory about
control of gene activity
• In bacteria many genes have an “operator” region
downstream of the promoter which affects transcription
• The promoter is the region that binds the RNA polymerase
• The operator region can bind a “repressor” protein. The
repressor protein blocks the movement of the polymerase
• If the action of the polymerase is blocked, transcription is shut
down (“repressed’)
Negative vs. Positive Control
Negative Control
• The gene is turned
on unless the
repressor binds to
the operator
• Since the repressor
turns off the operon,
control is negative
Positive Control
• The gene is on, and
transcription occurs
• If an activator protein
binds, it will facilitate
the binding of the
polymerase,
increasing the rate of
transcription
The trp operon
• Tryptophan is a necessary nutrient. The common intestinal
bacterium E. coli will use tryptophan when it is available from
its environment, but will need to produce tryptophan when it
isn’t available.
• E. coli makes tryptophan in a 3 step metabolic pathway from
precursor molecules. The pathway requires 3 enzymes.
• The genes for the enzymes are adjacent to each other on the
chromosome, and are transcribed in a single mRNA with 5
start codons and 5 stop codons
• The operon is regulated by a single promoter and operator
• The genes will be transcribed unless tryptophan is present
• When tryptophan is present it binds to the repressor and
changes its shape, allowing it to bind to the operator region to
turn the gene off
The trp operon
Repressible Negative Control
• The gene is generally turned on, but can be repressed (turned
off) by the binding of a repressor protein
• The repressor protein is present in the cell, but in an inactive
form. The repressor will become activated by the binding of a
corepressor molecule
• The corepressor may be the end product of the enzymatic
reaction that the operon codes for.
• In the case of the trp operon, tryptophan is the end product of
the metabolic pathway and also the corepressor.
• When the cell has an abundance of tryptophan, the cell will shut
off the mechanism for making more tryptophan
Repressible negative control
Trp Feedback Inhibition
The trp Pathway
Precursor
1st
Intermediate
2nd
Intermediate
Tryptophan
• The tryptophan
pathway consists of 3
enzymatic reactions
• The end product,
tryptophan, is
produced from an
organic precursor with
2 intermediates
• The 3 enzymes are
made from
polypeptides coded for
on 5 genes transcribed
on a single mRNA
The trp Pathway
Precursor
1st
Intermediate
2nd
Intermediate
• The first enzyme in
the pathway has an
allosteric site
• The end product,
tryptophan, can bind
to the allosteric site
of enzyme 1 and
inhibit its activity
(“allosteric inhibition”)
Tryptophan
Inducible Negative Control
• Inducible = able to be turned on
• Negative = controlled by the binding of a
repressor
• This type of control is characteristic of genes that
are usually not needed. The repressor protein is
usually bound to the operator, blocking
transcription
• If an inducer molecule binds to the repressor it
will detach from the operator, turning the gene
on
The lac pathway
• E. coli can digest lactose
(a disaccharide) into the
2 simple sugars glucose
and galactose.
• Once digested, the
simple sugars are
metabolized through
normal pathways
• The process involves 3
enzymes, all transcribed
from one operon on a
single mRNA
• There is no real
advantage to digesting
lactose if the cell already
has glucose available.
It’s more economical to
metabolize glucose till
the cell runs out, then
switch to lactose
metabolism
• But there is no point to
making enzymes for
lactose digestion if there
is no lactose available
The lac operon
• The lac operon is an example of inducible
negative control. The gene is turned off
unless the bacteria have lactose available
as a food source.
• The inducer molecule is an isomer of
lactose (called allolactose). When lactose
is present the operon becomes activated
The lac operon
The lac operon - Details
Lactose or Glucose?
• Glucose is easier for E. coli to metabolize than lactose, so if
both foods are available the cells will use the glucose first.
• The lac genes will be turned on, but transcribed at a low rate
• The rate will be increase by the binding of an activator protein
(positive control) only when glucose is scarce
• When glucose is plentiful, the cells metabolic rate will be high.
The cells content of ATP will be high, and the cell will have
very little AMP. The content of AMP will determine the rate of
transcription. The binding of AMP to the activator protein
changes its shape, allowing it to bind to the the operator
region and facilitate the activity of the polymerase
The role of AMP, ADP, and ATP
• The energy currency of the cell is ATP. It is produced by
phosphorylating ADP, much as ADP is produced by
phosphorylating AMP
• The mechanisms for phosphorylation were discussed in detail
in our Bioenergetics unit, but for the sake of review:
• Substrate level phosphorylation (in glycolysis)
• Chemiosmotic phosphorylation (in the electron transport chain)
• A cell with an abundance of food will have produced a large
quantity of ATP. This means that the quantity of AMP and ADP
will be low since almost all of it will have been phosphorylated
• A cell with a high content of AMP is starving, because it has
used up its available ATP and hasn’t been able to make more
cAMP
• When the cell has a large quantity of AMP (again, the cell
must be starving because its ATP is used up) some of that AMP
is modified. Technically, the phosphate at the 5’ position
forms a second bond to the pentose at the 3’ position –
basically a phosphodiester bond self contained within a single
nucleotide. Because this creates a ring structure the AMP is
now “cyclic” AMP (cAMP)
• Don’t get too hung up on the details. cAMP serves as a
signaling molecule, and the signal it’s sending is starvation.
cAMP means the cell is hungry, and because it’s hungry it
needs to turn on some alternative pathways to metabolism
Positive Control
Gene Regulation Videos
• Gene Regulation
• http://www.youtube.com/watch?v=3S3ZOmleAj0&list=PLFCE4D99C4124A27A
• Alternative Approaches to Molecular Biology
• https://www.youtube.com/watch?v=TnpCMgtDPgk&list=PLF83B8D8C87426E44
• The lac operon
• https://www.youtube.com/watch?v=2TL8rY9Rc_A&list=PLF83B8D8C87426E44
Rationale
• The central dogma of molecular genetics states that genes are
molecules of
that must be transcribed into
molecules of
which then are translated into
• The process of transcription is directed by an enzyme called
• Translation is directed by particles of RNA and protein called
which bind to the
• Molecules of
then align to codons,
matching the appropriate
to the code
• The ribosome then catalyzes the formation of
bonds between the
forming a
polypeptide
Regulation
• Economy demands that genes are only active when the cell
need the proteins that they code for. If there is an
accumulation of the end product of an enzymatic pathway, it
benefits the cell to turn the gene (on/off)
• It is most economical to exert control at the level of
(transcription/translation) by blocking the activity of the
(ribosomes/mRNA/tRNA/RNA polymerase) because the cell
will not need to bear the expense of producing
(ribosomes/mRNA/tRNA/RNA polymerase)
Fundamentals
• The promoter region is the area where
binds, allowing
• The operator region overlaps the promoter on the
downstream end, acting as a binding site for a repressor
protein. Binding of the repressor (allows/prevents) binding of
the RNA polymerase, (preventing/promoting) transcription
• Operons that use repressor proteins are under
(positive/negative) control because the repressor turns the
gene (on/off)
Repressible vs Inducible
The repressor protein is active when it binds to the operator.
Some repressors are already the proper shape to bind actively,
others only become active when also bound to an allosteric
molecule.
• An (inducer/corepressor) will (activate/inactivate) the repressor,
causing it to attach to the operator region, turning the gene
(on/off)
• An (inducer/corepressor) will (activate/inactivate) the repressor,
causing it to detach from the operator region, turning the gene
(on/off)
Feedback systems
• Negative feedback occurs when an accumulation of a
molecule (often the end product of a pathway) stimulates the
pathway to become (more/less) active
• The result of negative feedback is (amplification/homeostasis)
• An accumulation of tryptophan (increases/decreases) the
need for more tryptophan. The tryptophan will act as (an
inducer/a corepressor), binding to a repressor protein making
the repressor (active/inactive)
• The repressor will then (attach to/detach from) the operator
region, turning the operon (on/off) by (allowing/blocking)
transcription
Trp pathway feedback loop
• Tryptophan also exerts negative feedback control directly on
the metabolic pathway. Excess tryptophan will bind to the
allosteric site of the first enzyme in the trp synthesis pathway.
The trp acts as an allosteric (activator/inhibitor), changing the
shape of the enzyme to make it become (capable/incapable)
of binding to the substrate
• Exerting control at the enzyme level allows (faster/slower)
response and is (easier/more difficult) to reverse once
tryptophan levels diminish
Identify all parts of the system
The lac Operon
• E. coli can use either glucose (a monosaccharide) or lactose (a
disaccharide) for energy. If both food sources are available
the cell benefits by using the (glucose/lactose) first because it
doesn’t need to be digested.
• If lactose is available, the lac operon needs to be turned
(on/off) by (attaching/detaching) the repressor protein. This is
accomplished by binding allolactose to the repressor. The
allolactose acts as (an inducer/a corepressor)
• If glucose is available glucose will be metabolized, resulting in
an increase in the amount of (AMP/ATP) and a decrease in the
amount of (AMP/ATP)
• A “hungry” cell will have (high/low) quantities of cAMP which
will bind to the Activator Protein (increasing/decreasing) the
rate of transcription for the lac operon
Identify all parts of the system
Concept Check 18.1, page 356
1. How does the binding of the trp corepressor and the lac
inducer to their respective repressor proteins alter repressor
function and transcription in each case?
2. Describe the binding of RNA polymerase, repressors, and
activators to the lac operon when both lactose and glucose
are scarce. What is the effect of these scarcities on the
transcription of the lac operon?
3. A certain mutation in E. coli changes the lac operator so that
the active repressor cannot bind. How would this affect the
cell’s production of b-galactosidase? (b-galactosidase is
enzyme 1 of the lac pathway)