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Gene Regulation
Turning genes ON and OFF
All cells in an organism contain the
same DNA, so why are the cells
different?
BECAUSE, they contain and are
made up of different proteins.
BECAUSE, different genes are
transcribed in the different cells so
different mRNA is transcribed for
protein synthesis.
Genes are turned on and off. When a gene
is on, it is expressed meaning it is
transcribed. The DNA must unzip in order
for transcription—it just doesn’t have to
unzip entirely!
All cells have “housekeeping”
genes. These genes code for
proteins necessary for metabolism,
structure, and synthesis.
Some genes are tissue-specific
and are only expressed in certain
types of cells or tissues.
HOW do genes turn ON and OFF?
To conserve ENERGY, it is most
efficient to control this at the
transcription level. Why transcribe
mRNA that is not needed by the cell?
Transcription factors: These bind
to the DNA. Activators &
Repressors.
This grey line will
represent DNA
throughout this
lesson.
Genes that are regulated by the same
repressor or activator have the same
or closely related regulatory
sequences.
DNA regulatory sequences are
tissue-specific.
• Muscle-specific genes all
have a particular sequence
in their DNA to which a
muscle-specific transcription
factor binds and activates
expression of these genes.
• Nerve-specific genes have a
DIFFERENT sequence in the
DNA and different
transcription factors.
• These DNA base sequences
lie “upstream” from the base
sequences that will be
transcribed when the gene is
expressed.
More about those DNA sequences
DNA-Binding domains have been
conserved throughout evolution
• Transcription factors
are proteins
• They have distinct
regions with specific
functions
• One region is the
DNA-binding
domain—it IS a
region of the protein
that is the
transcription factor
Often the domain is an α-helix
This red thing,
that looks like a
stick of
dynamite, is
just another
representation
of the α-helix.
These are the 2 most common DNAbinding domains. If you can name
them & explain their function in an
essay, it’s often worth a point!
The binding domains of repressors
and activators contact specific
nucleotide sequences in DNA.
The regulatory region of a gene
has many binding sites upstream
from the promoter.
• Often these sites are near
the promoter, BUT they
can be distant as well.
• The nucleotide sequence
to which a positive
activator binds is called
an activator site.
• The nucleotide sequence
to which a negative
repressor binds is called
a repressor site.
Repressors prevent transcription by
being a “roadblock” so RNA
polymerase cannot bind to the DNA
at the promoter.
When a repressor is NOT bound,
RNA polymerase can bind to the
promoter and transcription begins.
Activators help RNA polymerase bind
to the DNA. Very active genes have
several RNA polymerases transcribing
them.
The structure and function of the
transcription factor can be altered
when other molecules bind to them.
INDUCERS
• Small molecules,
INDUCERS, can bind
to a transcription
factor [repressor in
this picture], cause a
conformational shape
change and decrease
its ability to bind DNA.
• This causes the
repressor to abandon
the DNA and
transcription is no
longer blocked!
KNOW THIS WELL!
OPERONS
• An OPERON is a
group of genes that
are transcribed
together.
• They are turned ON
or OFF as a unit.
• These 3 genes are
needed to metabolize
lactose.
• The repressor site of
an operon is called an
operator
These genes are only
turned ON if lactose is
present. Why waste
energy synthesizing
proteins that serve NO
function in the absence
of lactose?
Lactose  glucose + galactose
These genes code for the enzymes
that metabolize the above reaction!
Here the lac repressor is bound to the repressor
site, called the operator [since it controls the
operon]. It is the “roadblock” that blocks the
initiation of transcription at the promoter.
Allolactose is an INDUCER
• When lactose is
present, some of it
exists as an isomer
called allolactose.
• Allolactose acts as an
inducer.
• It binds to the Lac
Repressor, causing a
conformational shape
change.
This is called negative regulation of
the lac operon
• The change in shape,
causes the Lac
Repressor to release
the DNA so
transcription is no
longer blocked.
• RNA polymerase can
now bind to the
promoter and
transcription of the
lactose operon can
begin.
Once more, with feeling!
• IN THE ABSENCE OF LACTOSE, it is
normal for the lac repressor to be
bound to the operator.
• When lactose is present, it’s isomer—
allolactose—acts as an inducer.
• It binds to the repressor, changing its
shape.
• This causes the repressor to release
the DNA
• RNA polymerase can now bind to the
promoter so transcription of the 3 genes
comprising the operon can commence!
• Synthesis of the 3 proteins necessary
for the breakdown of lactose are now
manufactured and lactose can be
utilized by the cell.
This process is called NEGATIVE
REGULATION OF E. coli
LACTOSE METABOLISM
ACTIVATORS
• An activator steps up
the transcription rate
• More of the lactose
enzymes can be
transcribed at once—
assembly line style!
This process is POSITIVE Regulation of
the Lac Operon!
Catabolite Activator Protein: CAP
• CAP is the name of the
catabolite activator protein
• It needs cyclic AMP
[cAMP; ATPADPAMP]
bound to it before it can
have the proper shape in
order to bind to the
activator site of the DNA.
• Once bound, it
INCREASES the ability of
RNA polymerase to bind to
the promoter and
transcribe the genes
This is hyper drive!
The cAMP-CAP complex is very
attractive to RNA polymerase!
• Notice that the cAMPCAP complex contacts
BOTH the DNA at the
activator site AND the
RNA polymerase.
• This contact makes the
RNA polymerase have
MORE attraction for the
DNA, stepping up the rate
of transcription of the
operon.
What is the purpose?
• With this regulation, cells
are very E efficient!
• These cells would rather
use glucose as a food
source.
• That’s why it is normal for
the repressor to be in
place.
• As long as glucose is
available, the cell won’t
metabolize lactose.
• Glucose yields more
energy to the cells than
lactose.
Glucose helps the cells grow! As it is
used up, the number of dividing cells
stabilizes in the population.
Glucose availability regulates
cAMP production.
• These are bacterial
cells—glycolysis is
their only source of
ATP.
• When the glucose is
gone, ATP production
ceases.
• ATPADPAMP
SO, the concentration
of the cyclic form of
AMP will increase
What if both glucose and lactose
are present?
• Cells will use glucose first
until gone!
• cAMP production
increases as a result
• cAMP binds to the CAP
and the activator
complex, in turn, causes
RNA polymerase to crank
up the rate of
transcription of the lac
operon
• The enzymes necessary
to utilize lactose as an
energy source are
QUICKLY manufactured!
Explain this!
Negative control is caused by a repressor, which
can be affected by an inducer. Positive control
is caused by and activator. Repressors,
inducers and activators are transcription factors.
Which transcription factors are
bound to the DNA when the
different sugars are present?
No lactose? The repressor is
bound.
Lactose present? The allolactose inducer
causes the bound repressor to change shape
and release the DNA. The “roadblock” is
removed. Transcription begins.
Only lactose present? The absence of
glucose causes the cAMP level to rise and
allows the binding of the activator [cAMPCAP complex]. RNA polymerase’s affinity
for DNA is enhanced.
HOW IS THIS DIFFERENT IN
EUKARYOTES?
• Gene expression in eukaryotes has two main
differences from the same process in
prokaryotes.
• First, the typical multicellular eukaryotic
genome is much larger than that of a
bacterium.
• Second, cell specialization limits the
expression of many genes to specific cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The estimated 35,000 genes in the human
genome includes an enormous amount of
DNA that does not program the synthesis
of RNA or protein.
• This DNA is elaborately organized.
– Not only is the DNA associated with protein to
form chromatin, but the chromatin is organized
into higher organizational levels.
• Level of packing is one way that gene
expression is regulated.
– Densely packed areas are inactivated.
– Loosely packed areas are being actively
transcribed.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Chromatin structure is based on
successive levels of DNA
packing
• While the single circular chromosome of
bacteria is coiled and looped in a complex,
but orderly manner, eukaryotic chromatin is
far more complex.
• Eukaryotic DNA is precisely combined with
large amounts of protein.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Chromatin structure is based on
successive levels of DNA
packing
• During interphase of the cell cycle,
chromatin fibers are usually highly
extended within the nucleus.
• During mitosis, the chromatin coils and
condenses to form short, thick
chromosomes.
• Eukaryotic chromosomes contain an
enormous amount of DNA relative to their
condensed length.
– Each human chromosome averages about 2 x
108 nucleotide pairs.
– If extended, each DNA molecule would be
about 6 cm long, thousands of times longer
than the cell diameter.
– This chromosome and 45 other human
chromosomes fit into the nucleus.
– This occurs through an elaborate, multilevel
system of DNA packing.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Histone proteins are responsible for the
first level of DNA packaging.
– Their positively charged amino acids bind
tightly to negatively charged DNA.
– The five types of histones are very similar from
one eukaryote to another and are even present
in bacteria.
• Unfolded chromatin has the appearance of
beads on a string, a nucleosome, in which
DNA winds around a core of histone
proteins.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The beaded string seems to remain
essentially intact throughout the cell cycle.
• Histones leave the DNA only transiently
during DNA replication.
• They stay with the DNA during
transcription.
– By changing shape and position, nucleosomes
allow RNA-synthesizing polymerases to move
along the DNA.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• As chromosomes enter mitosis the beaded
string undergoes higher-order packing.
• The beaded string coils to form the 30-nm
chromatin fiber.
• This fiber forms looped domains attached
to a scaffold of nonhistone proteins.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In a mitotic
chromosome,
the looped
domains
coil and fold to
produce the
characteristic
metaphase
chromosome.
• These packing
steps are highly
specific and
precise with
particular genes
located in the
same places. Fig. 19.1
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Chromatin
• Interphase chromatin is generally much less
condensed than the chromatin of mitosis.
– While the 30-nm fibers and looped domains
remain, the discrete scaffold is not present.
– The looped domains appear to be attached to the
nuclear lamina and perhaps the nuclear matrix.
• The chromatin of each chromosome occupies
a restricted area within the interphase
nucleus.
• Interphase chromosomes have areas that
remain highly condensed, heterochromatin,
and less compacted areas, euchromatin.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
DNA packing
Many binding sites
• The regulatory region
of a gene is very
complex in
multicellular
organisms
• Binding sites for
MANY transcription
factors
• Regulatory regions
contain 1,000s of
base pairs of DNA
DNA looping
• DNA is flexible and
can form loops
DNA looping
• This allows
transcription factors to
interact over long
distances
DNA looping
• Hence the RATE of
transcription and the
amount of mRNA and
protein synthesis is
determined by these
interactions
Transcription factor binding in
eukaryotes; influences
• The interactions of
transcription factors
with one another or
with DNA can be
influenced by signals
– From within the cell
– From outside the cell
• Signals are integrated
at the promoter
Transcription factor—low nutrients
• Nutritional state of a
cell may affect the
activity of various
transcription factors
• Absence of a nutrient
may signal a
particular repressor to
bind to an operator
and turn off the
expression of a gene
Transcription factor—high nutrients
• For a different gene,
the presence of the
same nutrient may
signal a transcription
factor to bind in the
regulator region and
stimulate a high level
of transcription
• What do we call this
type of transcription
factor?
Transcription factor—time of day
• At a particular time of day
[lunch?] a second
transcription factor may
bind to augment
transcription to an even
higher level
• This type of control is an
internal clock regulation
and would be
independent of the
availability of food
• What do we call this type
of transcription factor?
Transcription factor--Tissue specific
• Transcription of the gene
that was turned on by
nutrient availability and
time of day in cell A may
be turned off completely
by the presence of a
repressor in cell B
• Interaction of transcription
factors with
– DNA, other factors and the
environment can
dramatically alter the
expression of a gene
Turning on a gene
This will cost more ENERGY!
Not the best plan!
Post-transcriptional control
Mechanism Overview
• These mechanisms act AFTER mRNA has been
synthesized
– Costs the cell more Energy!
• The mechanisms affect the amount of protein made by
the cell
Tissue-specific mRNA splicing
• The type of proteins made by a cell can be
regulated by splicing the precursor mRNA
in different ways
• Some mRNAs are spliced in a tissuespecific manner so that two different
proteins are made in two different types of
cells
Thyroid cells splice together exons
1,2,3 and 4 of the gene to form an
mRNA that is translated into the
calcitonin protein
Brain cells splice together exons
1,2,3,5, and 6 from the same mRNA
to form the neuropeptide CGRP
mRNA Stability
• The poly-A tail on
mRNA is important for
its stability.
• If the poly-A tail is
removed, then RNA is
rapidly degraded and
less protein is made
mRNA stability
• Proteins that bind to
mRNA can influence
survival of the poly-A
tail and hence
influence the amount
of protein translated
form the mRNA
RNA compartmentalization
• mRNAs can be
transported to specific
sections of the cell in
which the translated
products of the mRNA
will be used
• Some mRNAs are
sequestered in
sections of a cell until
they are needed
Translational control
• Alters the rate at
which ribosomes bind
to a mRNA and make
protein
• A tissue-specific
blocking protein may
bind to the RNA and
inhibit the binding of
ribosomes
Control of translation
Summary
• A gene is transcribed into mRNA in the
nucleus
• mRNA is also processed in the nucleus by
splicing and adding the poly-A tail
• Splicing influences the ultimate product of
the mRNA
• IN THE CYTOPLASM, the availability of
mRNA to ribosomes and the presence of
molecules that either protect or degrade
the mRNA will influence the amount of
protein made.
RNA processing
Antibody structure
• Antibodies are proteins
that recognize invaders
[antigens]
• Synthesized by cells in
which DNA
rearrangement produces
a functional antibody
gene
• Composed of 4
polypeptide chains
– 2 heavy (H) & 2 light (L)
– H is composed of 3
regions: variable, joining
and constant: V,J & C
– Each antibody recognizes
and binds to one specific
antigen
– We need a great diversity
of antibodies to protect
against a variety of
invaders [pathogens]
What are those?
Antibody light chain gene
• The genome contains several copies of
the different parts of the antibody genes.
• In undifferentiated cells, each gene for the
L of antibodies includes
– 100s of different V regions
– Several J regions
– One or more different C regions
• The genes for the H chains are organized
similarly
Antibody specificity created by
gene rearrangement
• During differentiation of immune cells that
are destined to produce antibodies, the
antibody genes are rearranged via
recombination to produce functional
antibody genes
• Rearrangement of the gene occurs by
joining together one segment from each of
the 3 regions of the gene and by deleting
the other, extra DNA
The number of combinations of
segments from the 3 regions is
tremendous
Which parts
are exons?
Which parts
are introns?
Antibody specificity created by
gene rearrangement
• Each L chain protein produced by the cell
contains one V, J and C region
• The H chain is produced by a similar
rearrangement process
• The two light chains are identical
• The two heavy chains are identical
• Each combination of a light chain and a
heavy chain can recognize one specific
antigen
1000s of antibodies on patrol
• Recombination of antibody gene DNA segments
can occur at many different sites but always
occur between V-J and J-C regions
• The antibody expressed by a cell is determined
by the combination of V, J and C regions that
are NOT deleted.
• This creates the potential for each cell to make
any one of 1000s of different antibody
molecules, each of which can recognize a
different antigen
Recombinations occur between
V-J and J-C regions
Each developing antibodyproducing cell has the potential to
form a unique antibody
• Each cell makes only
one type of antibody
because its genes
undergo
rearrangement early
in the differentiation of
the cell
• The cell is thereafter
committed to
produce only one
type of antibody
Gene amplification
• Certain regions of
DNA can undergo
EXTRA rounds of
DNA replication
Gene amplification
• This process is called
amplification
Gene amplification
• It creates MORE
copies of DNA and
hence the potential to
make more RNA
Gene amplification
• And you know what
that means…
• …more proteins!
Gene amplification of rRNA genes
in amphibian eggs
• In amphibian eggs,
genes for rRNA
become highly
amplified
Gene amplification of rRNA genes
in amphibian eggs
• The amplification, in
this case, begins with
recombination
between repeated
copies of the rRNA
genes, resulting in the
production of circular
DNA molecules
containing the rRNA
genes
Gene amplification of rRNA genes
in amphibian eggs
Far more efficient that duplicating
DNA the old fashioned way!
• These small, circular
DNA molecules
replicate to produce
1000s of copies of the
rRNA genes
• This allows the egg to
quickly make many
ribosomes which are
needed for protein
synthesis after
fertilization
Gene amplification—role in
resistance to cancer drug
• Gene amplification
can also be important
in disease
• Methotrexate is a
drug used to treat
cancer patients
• Tumor cells that are
rapidly synthesizing
DNA are more
damaged by the drug
than normal cells are
Gene amplification—role in
resistance to cancer drug
• Methotrexate inhibits
the activity of
dihydrofolate
reductase (DHFR), an
enzyme needed for
synthesis of
deoxyribonucleotides
• A cancer cell may
become resistant to
methotrexate if
amplification of the
DHFR gene occurs
Gene amplification—role in
resistance to cancer drug
• Multiple copies of the
gene encoding DHFR
allow enough enzyme
to be made for the
cell to continue to
grow in the presence
of methotrexate
Gene amplification—role in
resistance to cancer drug
Importance of gene regulation
• The amount and time of gene expression can be
regulated at any one of several steps between
the DNA and the final functional gene product