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XII. Gene Regulation
- Overview:
All cells in an organism contain the same genetic information; the key to tissue
specialization is gene regulation – reading some genes in some cells and other
genes in other cells.
Also, organisms can respond to their environment at a genetic level, so there
must be a way for the environment to stimulate or repress the action of
certain genes.
And changes occur through time, creating developmental changes. We will
look at how gene expression is regulated in these cases.
- Overview:
-Some Terminology:
- some enzymatic genes are only turned on if the substrate is present; this is
an inducible system and the substrate is the inducer. Obviously, this is highly
adaptive, as the cell saves energy by only producing the enzyme when it is
needed.
- Overview:
-Some Terminology:
- some enzymatic genes are only turned on if the substrate is present; this is
an inducible system and the substrate is the inducer. Obviously, this is highly
adaptive, as the cell saves energy by only producing the enzyme when it is
needed.
- some enzymes are on all the time, and are only turned off if a compound
(often the product of the metabolic process they are involved with) is present.
This is a repressible system, and the compound is the repressor. This is also
adaptive, and the cell saves on enzymes if the product is already present.
- Overview:
-Some Terminology:
- some enzymatic genes are only turned on if the substrate is present; this is
an inducible system and the substrate is the inducer. Obviously, this is highly
adaptive, as the cell saves energy by only producing the enzyme when it is
needed.
- some enzymes are on all the time, and are only turned off if a compound
(often the product of the metabolic process they are involved with) is present.
This is a repressible system, and the compound is the repressor. This is also
adaptive, and the cell saves on enzymes if the product is already present.
- Constitutive genes are on all the time.
XII. Gene Regulation
A. The lac Operon in E. coli
XII. Gene Regulation
A. The lac Operon in E. coli
- When lactose is present, E. coli produce three enzymes involved in lactose
metabolism. Lactose is broken into glucose and galactose, and galactose is
modified into glucose, too. Glucose is then metabolized in aerobic respiration
pathways to harvest energy (ATP). When lactose is absent, E. coli does not
make these enzymes and saves energy and amino acids. How do they KNOW?
:)
XII. Gene Regulation
A. The lac Operon in E. coli
As you remember, an “operon” was a
region of genes that are regulated as a
unit – it typically encodes > 1 protein
involved in a particular metabolic
pathway.
XII. Gene Regulation
A. The lac Operon in E. coli
As you remember, an “operon” was a
region of genes that are regulated as a
unit – it typically encodes > 1 protein
involved in a particular metabolic
pathway.
XII. Gene Regulation
A. The lac Operon in E. coli
Lac Y - permease – increases absorption of lactose
XII. Gene Regulation
A. The lac Operon in E. coli
Lac Y - permease – increases absorption of lactose
Lac Z – B-galactosidase – cleaves lactose into glucose and galactose
XII. Gene Regulation
A. The lac Operon in E. coli
Lac Y - permease – increases absorption of lactose
Lac Z – B-galactosidase – cleaves lactose into glucose and galactose
Lac A – transacetylase – may code for enzymes that detoxify waste production of
digestion.
XII. Gene Regulation
A. The lac Operon in E. coli
1960 – Jacob and Monod proposed that this was an inducible system
because the presence of the substrate INDUCES transcription.
Promoter
Repressor
Gene
Repressor
Operator
RNA Poly
XII. Gene Regulation
A. The lac Operon in E. coli
1960 – Jacob and Monod proposed that this was an inducible system
because the presence of the substrate INDUCES transcription.
LACTOSE
XII. Gene Regulation
A. The lac Operon in E. coli
The binding of lactose changes the shape of
the repressor (allosteric reaction) and it
can’t bind to the operator.
1960 – Jacob and Monod proposed that this was an inducible system
because the presence of the substrate INDUCES transcription.
LACTOSE
XII. Gene Regulation
A. The lac Operon in E. coli
Mutant analyses confirmed these results:
XII. Gene Regulation
A. The lac Operon in E. coli
Mutant analyses confirmed these results:
XII. Gene Regulation
A. The lac Operon in E. coli
Mutant analyses confirmed these results:
XII. Gene Regulation
A. The lac Operon in E. coli
Mutant analyses confirmed these results:
Curiously, there are only about 10 repressor
molecules in each cell and they were not actually
isolated and identified for 6 years (Gilbert).
XII. Gene Regulation
A. The lac Operon in E. coli
But it is even more complicated… if glucose AND lactose are present, the
operon is OFF. This is adaptive, because it’s glucose the cell needs. If
glucose is present, there is no need to break lactose down to get it. BUT
HOW?
XII. Gene Regulation
A. The lac Operon in E. coli
But it is even more complicated… if glucose AND lactose are present, the
operon is OFF. This is adaptive, because it’s glucose the cell needs. If
glucose is present, there is no need to break lactose down to get it. BUT
HOW?
This involves a repressible pathway.
XII. Gene Regulation
A. The lac Operon in E. coli
Within the promoter, there is a binding site for Catabolic Activating Protein
– basically a “transcription factor”. CAP needs to bind in order for the RNA
Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric
reaction so it can bind the promoter.
, lactose present
XII. Gene Regulation
A. The lac Operon in E. coli
Within the promoter, there is a binding site for Catabolic Activating Protein
– basically a “transcription factor”. CAP needs to bind in order for the RNA
Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric
reaction so it can bind the promoter. So, the binding of CAP stimulates
transcription.
, lactose present
XII. Gene Regulation
A. The lac Operon in E. coli
When Glucose is present, the concentration of c-AMP declines, it does not
bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does
not bind either and the genes are off.
, lactose present
CAP
REPRESSOR
XII. Gene Regulation
A. The lac Operon in E. coli
When Glucose is present, the concentration of c-AMP declines, it does not
bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does
not bind either and the genes are off.
So, the lac operon is regulated first by the presence/absence of glucose; the
needed nutrient…and then by the presence of lactose, which could be
metabolized to produce glucose if necessary.
XII. Gene Regulation
A. The lac Operon in E. coli
B. The trp Operon in E. coli
XII. Gene Regulation
A. The lac Operon in E. coli
B. The trp Operon in E. coli
Tryptophan is an amino acid that can be synthesized by tryptophan
synthetase. This gene and its partners are only ON if tryptophan is absent.
The presence of tryptophan represses the production of these enzymes
(repressible system).
B. The trp Operon in E. coli
If trp is absent, the
repressor can’t bind to
the operator…
transcription proceeds..
B. The trp Operon in E. coli
If trp is present, it binds
to the repressor,
changing the
repressor’s shape so
that it can now bind to
the operator and inhibit
RNA poly binding.
B. The trp Operon in E. coli
Secondary Regulation
Actually, when trp is present,
B. The trp Operon in E. coli
Secondary Regulation
ACTUALLY, TRANSCRIPTION
ALWAYS PROCEEDS A LITTLE
BIT…UP TO THE REGION CALLED
THE “ATTENUATOR”…
B. The trp Operon in E. coli
Secondary Regulation
ACTUALLY, TRANSCRIPTION
ALWAYS PROCEEDS A LITTLE
BIT…UP TO THE REGION CALLED
THE “ATTENUATOR”…
B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the mRNA; the 3-4 loop causes termination
of transcription.
B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the mRNA; the 3-4 loop causes termination
of transcription.
Because translation occurs as soon as
m-RNA is produced, ribosomes jump
on and begin to read the strand…
there are two trp codons at the
beginning of the sequence.
B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the mRNA; the 3-4 loop causes termination
of transcription.
Because translation occurs as soon as
m-RNA is produced, ribosomes jump
on and begin to read the strand…
there are two trp codons at the
beginning of the sequence.
If trp is present, the ribosome zooms
along (incorporating trp) and it
occupies the 2 region… region 3 is
free to bind with 4 and the
termination loop forms…
B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the mRNA; the 3-4 loop causes termination
of transcription.
Because translation occurs as soon as
m-RNA is produced, ribosomes jump
on and begin to read the strand…
there are two trp codons at the
beginning of the sequence.
If trp is present, the ribosome zooms
along (incorporating trp) and it
occupies the 2 region… region 3 is
free to bind with 4 and the
termination loop forms…
If low trp, then ribosome stalls; region
3 bind to 2, no termination loop
forms, and transcription of the genes
proceeds…Translation of the genes begins at start codons downstream…
XII. Gene Regulation
A. The lac Operon in E. coli
B. The trp Operon in E. coli
C. Regulation in Eukaryotes
XII. Gene Regulation
A. The lac Operon in E. coli
B. The trp Operon in E. coli
C. Regulation in Eukaryotes
- higher levels of packaging, intron-exon structure, and the need for tissue
specialization makes regulation in eukaryotes far more complex than
responding to environmental cues.
XII. Gene Regulation
A. The lac Operon in E. coli
B. The trp Operon in E. coli
C. Regulation in Eukaryotes
- higher levels of packaging, intron-exon structure, and the need for tissue
specialization makes regulation in eukaryotes far more complex that
responding to environmental cues.
1. Histone Regulation
- Core DNA, bound to histones, is OFF. Only “linker DNA”, between histones,
is even accessible to RNA polymerases. So, binding DNA to histones is the
first way to shut it off.
C. Regulation in Eukaryotes
1. Histone Regulation
- Three ways to reveal DNA
“chromatin remodeling”
C. Regulation in Eukaryotes
1. Histone Regulation
- Three ways to reveal DNA
“chromatin remodeling”
2. Methylation
- highly repetitive sequences
- imprinted genes
- Barr bodies
C. Regulation in Eukaryotes
1. Histone Regulation
- Three ways to reveal DNA
“chromatin remodeling”
2. Methylation
- highly repetitive sequences
- imprinted genes
- Barr bodies
Some proteins bind to the methylated cytosines, and may either recruit
repressors or interrupt transcription factor binding.
C. Regulation in Eukaryotes
1. Histone Regulation
2. Methylation
3. Promoters
- Several consensus sequences (TATA, CAAT, GGGCGG) appear in
combination in nearly all promoters and are required for basal levels of
transcription
C. Regulation in Eukaryotes
1. Histone Regulation
2. Methylation
3. Promoters
4. Enhancers/Silencers
Cis-acting elements on the same chromosome, which regulate a neighboring
gene.
They are somewhat like operators, in that they are binding sites for
transcription factors that can “up” or “down” regulate transcription.
However, they function ANYWHERE near the gene: before, within, or after
C. Regulation in Eukaryotes
1. Histone Regulation
2. Methylation
3. Promoters
4. Enhancers/Silencers
Cis-acting elements on the same chromosome, which regulate a neighboring
gene.
They are somewhat like operators, in that they are binding sites for
transcription factors that can “up” or “down” regulate transcription.
However, they function ANYWHERE near the gene: before, within, or after
They are not gene specific – they will enhance their neighbor
Silencers tend to reduce binding of the polymerase to the promoter.
C. Regulation in Eukaryotes
1. Histone Regulation
2. Methylation
3. Promoters
4. Enhancers/Silencers
These are the transcription factors that bind to enhancer and silencer regions
of the human metallothionien IIA gene promoter region!!
- What does having all these modifiers allow for?
Different proteins can silence or enhance
DNA Polymerase II binding. This may involve
the formation of a pre-initiation complex” of
proteins that allow the Poly II to bind, and can
even involve sequences far from the promoter
that loop – and influence RNA Poly II activity.
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
- These are the proteins that bind to DNA and influence transcription.
They have “binding domains” that bind DNA in particular ways.
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
- These are the proteins that bind to DNA and influence transcription.
They have “binding domains” that bind DNA in particular ways.
HTH = “helix-turn-helix”
One class of important
HTH TF’s contain specific
sequences of AA’s called a
homeodomain. This is
encoded by a 180 bp
region in it’s gene called a
homeobox. These
homeotic genes/proteins
are conserved across all
eukaryotes and are critical
to basic animal
development.
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
- These are the proteins that bind to DNA and influence transcription.
They have “binding domains” that bind DNA in particular ways.
“Zinc-Finger”: Zinc binds to two cysteine and
two histidine AA’s. The sequence between forms
A loop or “finger”, and the specific AA sequence
Binds specific DNA sequences…
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
We didn’t really know what
they did in vivo.
Biochemists have linked
other proteins to them,
however, making Zincfinger nucleases that cut
DNA at specific
sequences.
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
But Feb 18, 2015, Najafabadi et al. found this:
- “Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest
class of putative human transcription factors” (> 700 proteins)
- “C2H2-ZF proteins recognize more motifs than all other human
transcription factors combined.” (Highly variable, tough to study)
- “C2H2-ZF proteins bind specific endogenous retroelements
(EREs), ranging from currently active to ancient families. The
majority of C2H2-ZF proteins, also show widespread binding to
regulatory regions, indicating that the human genome contains an
extensive and largely unstudied adaptive C2H2-ZF regulatory
network that targets a diverse range of genes and pathways.”
- They stabilized Endogenous Retroviral Elements, and evolved to
regulate other genes, as well.
Najafabadi et al. 2015
READ THIS
Science Daily
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
- These are the proteins that bind to DNA and influence transcription.
They have “binding domains” that bind DNA in particular ways.
bZIP=“basic leucine zipper”: leucine AA’s in
Different chains dimerize and the leucines “zip”
The other alpha-helices bind specific DNA sequences
C. Regulation in Eukaryotes
4. Enhancers/Silencers
5. Transcription Factors
- These are the proteins that bind to DNA and influence transcription.
They have “binding domains” that bind DNA in particular ways.
- Then, the TF’s have other binding sites for
Proteins (like basal transcription factors) or
Other chemicals (like hormones)
C. Regulation in Eukaryotes
5. Transcription Factors
6. Alternate Splicing Pathways
- Many proteins can be made from the same gene, by splicing the m-RNA
differently. Humans have 20-30K genes, but several 100,000 proteins!
A calcium regulator in the thyroid
A hormone made in the brain
C. Regulation in Eukaryotes
6. Alternate Splicing Pathways
7. Controlling m-RNA stability
Existing tubulin units interact with
a new tubulin strand and
translation stalls, releasing RNAse
that cleave the m-RNA.
So tubulin is only made when free
tubulin units are not present.
C. Regulation in Eukaryotes
7. Controlling m-RNA stability
8. RNA Silencing
- Short pieces of RNA can bind to
DNA in the nucleus or m-RNA
in the cytoplasm and regulate
gene expression.
C. Regulation in Eukaryotes
7. Controlling m-RNA stability
8. RNA Silencing/Interference
- si-RNA (small interfering RNA):
Viral or retrotransopon origin
- mi-RNA (micro-RNA):
Produced by intronic, sequences,
or different genes in genome.
Have stem-loop structure.
C. Regulation in Eukaryotes
7. Controlling m-RNA stability
8. RNA Silencing/Interference
THEY BOTH are attacked by DICER
protein, which cuts them into
short ds-RNA molecules.
These complex with RNA-induced
Silencing Complex proteins
(RISC) that denature the RNA
and degrade the sense strand.
What is left is a strand that is
complementary to a specific
m-RNA molecule.
C. Regulation in Eukaryotes
7. Controlling m-RNA stability
8. RNA Silencing/Interference
If the ss-RNA is exactly
complementary to a m-RNA,
RISC cuts the m-RNA into
fragments (turning protein
synthesis OFF).
Or, if not exactly complementary,
then the RISC complex stays
attached, interrupting
ribosome binding and
translation.
C. Regulation in Eukaryotes
7. Controlling m-RNA stability
8. RNA Silencing/Interference
OR!
The ds-RNA gets complexed with
RNA-induced initiation of
transcription silencing
complex (RITS). These
denature the RNA, creating ssRNA that binds to DNA
promoters or large regions of
DNA.
This binding attracts chromatin
remodeling proteins that
methylates the histones,
causing it to coil into
herochromatin (Turning Genes
OFF).
The process of Gene Activity – in terms of a Gene
Making a Functional Protein – can be regulated at
every step of the process, from:
Gene availability and chromatin structure
Transcription
Transcript Processing
Translation
Post-translational Modification
Variation in patterns of regulation lead to
differences in expression between cells, and cell
specialization.