LECTURE 017v2 – Regulation of Gene Expression
1. REGULATION OF EUKARYOTIC GENE EXPRESSION
a. How does an organism express certain genes in certain cells at certain times
and why?
b. This is a fluorescence micrograph of the complex organization of the
chromatin in a eukaryotic chromosome of a developing salamander egg.
c. Part of the chromatin is packed into the main axis (white) of the
chromosome, while those parts that are being actively transcribed are
spread out in loops (red).
d. Would an efficient machine waste energy producing all proteins from all
genes at all times, even when not needed?
e. Both prokaryotes and eukaryotes must alter their patterns of gene
expression in response to changes in environmental conditions.
2. REGULATION OF GENE EXPRESSION
a. GENE EXPRESSION: the process by which DNA directs the synthesis of
proteins (or in some cases, just RNA’s).
b. GENE EXPRESSION: Overall process by which the information encoded in a
gene is converted into an observable phenotype.
c. Unicellular or multicellular, genes are turned on or off in response to the
environment.
d. How did E. coli respond to its environment and adjust its gene expression in
our lab?
3. REGULATION OF A METABOLIC PATHWAY
a. METABOLIC CONTROL OCCURS ON TWO LEVELS:
b. Feedback inhibition: Cells can adjust the activity of enzymes already
present. The activity of the first enzyme in the tryptophan pathway is
inhibited by the pathway’s end product. If tryptophan accumulates it shuts
down additional synthesis.
4. REGULATION OF A METABOLIC PATHWAY
a. 2ND: REPRESS EXPRESSION: Cells can adjust the production level of certain
enzymes by regulating the EXPRESSION OF GENES encoding the enzyme.
b. If the environment is providing all of the tryptophan the cell needs, the cell
stops making the enzymes that catalyze its synthesis – stops producing
mRNA's.
c. HOW? Genes are switched on or off by changes in the metabolic status of
the cell.
d. First – we need to understand operons.
5. Anatomy of an Operon
a. Operon: A unit of genetic function found in bacteria and phages, consisting
of a promoter, an operator, and a cluster of genes whose products function
in a common pathway
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6. Operator: In bacterial DNA, a sequence of nucleotides near the start of an operon
to which an active repressor can attach. The binding of the repressor prevents
RNA polymerase from attaching to the promoter and transcribing the genes of the
operon.
i. THE OPERATOR CONTROLS THE ACCESS OF RNA POLYMERASE TO THE
GENES.
7. PROMOTER: DNA sequence where RNA polymerase attaches and initiates
transcription (upstream portion of the gene). Determines which of the two strands
of the DNA helix is used as the template.
a. RNA polymerase: An enzyme that links ribonucleotides into a growing RNA
chain during transcription, based on complementary binding to nucleotides
on a DNA template strand.
8. Repressor. Regulatory Gene.
a. REPRESSOR: a PROTEIN that INHIBITS gene expression. (Here by binding to
the operator at the promoter.)
b. Repressors bind to DNA in or near the promoter.
c. Regulatory gene: A gene that codes for a protein, such as a repressor, that
controls the transcription of another gene or group of genes.
9. TYPES OF OPERONS and REPRESSORS
a. Repressible operon: transcription of genes is usually ON – but can be
REPRESSED by a small molecule (ex: corepressor). Example of repressible
operon: trp operon.
b. Inducible operon: transcription is usually OFF, but it could be INDUCED to
turn on by a small molecule (ex: inducer). Example of inducible operon: lac
operon.
c. Repressors- two ways to activate:
i. Are activated by a corepressor (to switch an operon OFF).
ii. Are inactivated by an inducer (to switch an operon ON).
10.
a.
b.
c.
d.
e.
11.
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OPERONS - START BASIC: trp operon in E. coli
The trp operon is responsible for the synthesis of tryptophan.
The trp operon is a “repressible” operon meaning its normal state of being is
“ON”.
TRYPTOPHAN ABSENT FROM ENVIRONMENT: repressor inactive, operon ON.
Coordinate control is an “on/off” switch called the operator.
This operon can be turned off by a repressor with a corepressor (needed to
activate “inactive repressor”).
trp operon in E. coli
a. TRYPTOPHAN PRESENT IN ENVIRONMENT: repressor activated by
corepressor tryptophan, (why is tryptophan the corepressor? It is the
SIGNAL that there is an ABUNDANCE OF TRYPTOPHAN in the environment
and is NOT NEEDED!) operon OFF, stop making tryptophan.
b. The binding of repressors is reversible.
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c. This is negative gene regulation.
d. NEGATIVE GENE CONTROL: Operons are switched off by the active form of
the repressor protein.
e. When tryptophan accumulates, it acts as a co-repressor, which binds to the
inactive repressor and activates it, and in turn, it attaches to the operator
and REPRESSES transcription.
f. The trp operon is said to be “repressible” because its transcription is usually
on, but can be inhibited (repressed).
12.
a.
b.
c.
d.
13.
INDUCIBLE OPERON:
a. Normal state off: Lactose present - An inducer, allolactose (isomer of
lactose… present when lactose is present), inactivates the repressor,
OPERON ON.
RNA polymerase produces mRNA.
i. If lactose is added to the cells surroundings, allolactose binds to the
lac repressor and alters its configuration, repressor cannot attach to
the operator.
ii. 1. Lactose present, therefore, allolactose present.
iii. 2. Allolactose, an inducer, inactivates repressor.
iv. 3. Inactive repressor allows RNA polymerase to transcribe genes for
lactose metabolism.
14.
a.
b.
c.
d.
e.
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Inducible operon – “lac operon”
The lac operon is responsible for the production of three enzymes involved in
metabolizing lactose (when present).
Lactose absent, repressor active, operon off.
Regulatory gene lac I codes for an allosteric repressor protein that can
switch off the lac operon by binding to the operator.
The lac repressor, in contrast to the trp repressor, is active by itself. How to
INACTIVATE? An inducer, allolactose (isomer of lactose when lactose is
present), inactivates the repressor.
POSITIVE GENE REGULATION
In positive gene regulation, regulatory protein (CAP) interacts DIRECTLY
with GENOME.
E. coli preferentially uses glucose even in the presence of lactose.
Enzymes for glucose metabolism (glycolysis!) are continuously present. Not
true with lactose..
POSITIVE GENE CONTROL: when a regulatory protein interacts directly
with the genome to switch transcription on.
LAC OPERON: Under dual control:
i. Negative control by the lac repressor which determines whether or not
transcription of the lac operon’s genes occurs at all. ON-OFF CONTROL.
ii. Positive control by CAP: controls the rate of transcription if the operon
is repressor free. VOLUME CONTROL
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15.
Inducible Operon, Positive Gene Control
a. Lactose present, glucose scarce: abundant lac mRNA synthesized.
b. When only lactose is available, only then does E. coli make enzymes for
lactose breakdown.
c. How does E. coli know?
d. 1. Cyclic AMP (cAMP) accumulates when glucose is scarce.
e. 2. High concentration of cyclic AMP (cAMP) activates CAP.
f. 3. ACTIVE CAP increases the rate of transcription of lactose metabolizing
enzymes.
16.
a.
b.
c.
d.
e.
f.
g.
h.
Positive Gene Regulation-Glucose Present, Lactose Present
The lac repressor (bound or not with allolactose) determines if lac genes are
transcribed.
CAP determines the RATE of transcription based on cAMP concentration.
1. Glucose present.
2. cAMP level low.
3. CAP inactivated.
4. RNA polymerase less likely to bind to lac promoter.
When glucose is present, cAMP is scarce, and CAP is unable to stimulate
transcription (cAMP actives cap in an allosteric regulatory fashion).
CAP helps to regulate other operons – possibly affecting more than 100
genes in E. coli.
17.
REGULATION OF EUKARYOTIC GENE EXPRESSION
a. DIFFERENTIAL GENE EXPRESSION: the expression of different sets of genes
by cells with the same genome.
b. Each cell expresses ~ 20% of their genes at any given time.
18.
REGULATION OF EUKARYOTIC GENE EXPRESSION
a. Gene expression is equated with transcription for both bacteria and
eukaryotes.
b. Important difference: eukaryotic DNA is PACKAGED INTO NUCLEOSOMES,
forming CHROMATIN
c. Bacterial: open
d. Eukaryotic: not accessible to regulatory proteins, transcriptional apparatus
19.
DNA to RNA to PROTEIN
a. Eukaryotic gene expression can be regulated at any stage:
b. All organisms must regulate WHICH GENES are expressed AT ANY GIVEN
TIME.
c. Unicellular or multicellular, GENES ARE TURNED ON AND OFF IN RESPONSE
TO THEIR INTERNAL AND EXTERNAL ENVIRONMENTS.
d. REGULATION OCCURS: Chromatin modification, initiation of transcription,
alternative RNA splicing, translation, polypeptide modification, mRNA
stability.
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20.
REVIEW DNA AND CHROMATIN STRUCTURE
a. Modification of chromatin structure is a distinctive feature of eukaryotic gene
regulation.
b. CHROMATIN STRUCTURE USUALLY HAS TO BE CHANGED TO ACTIVATE
EUKARYOTIC TRANSCRIPTION.
c. The chromatin in chromosomal regions that are not being transcribed or
replicated exists predominantly in the condensed 30 nm fiber form. In many
cases, the binding of the transcriptional apparatus is not possible, owing to
the position of the nucleosomes near the promoter. (see page 328-329 in
chapter 16).
21.
CHROMATIN AND EUKARYOTIC GENE EXPRESSION
a. BEFORE TRANSCRIPTION, CHROMATIN STRUCTURE CHANGES AND THE DNA
BECOMES MORE ACCESSIBLE TO TRANSCRIPTIONAL MACHINERY.
22.
a.
b.
c.
d.
23.
CHROMATID MODIFICATION AND GENE EXPRESSION
TWO TYPES OF CHROMATIN:
Heterochromatin: eukaryotic chromatin that remains highly compacted
during interphase and is generally not transcribed.
Euchromatin: The less condensed form of eukaryotic chromatin that is
available for transcription.
GENE REGULATION depends on location of gene’s promoter relative to DNA
attachment to the chromosome scaffold and altering the location of a gene’s
promoter relative to a nucleosome.
CHROMATID MODIFICATION AND GENE EXPRESSION
a. CHROMATIN REMODELING: the changing of nucleosome position along DNA.
b. A “nucleosome make-over.”
c. Remodeling ultimately leads to altered accessibility of transcription factors to
regulatory DNA.
24.
CHEMICAL MODIFICATIONS TO HISTONES PLAY A DIRECT ROLE IN THE
REGULATION OF GENE TRANSCRIPTION.
a. Histone: a small protein with a high proportion of positively charged amino
acids that binds to the negatively charged DNA and plays a key role in
chromatin structure.
25.
HISTONES AND EUKARYOTIC GENE EXPRESSION
a. CHROMATIN: Complex of DNA, histones, and non-histone proteins from
which eukaryotic chromosomes are formed.
b. Nucleosome: The structural unit of chromatin consisting of a disk shaped
core of eight histone proteins around which a 147 base pair segment of DNA
is wrapped twice.
26.
HISTONES AND EUKARYOTIC GENE EXPRESSION
a. HISTONE MODIFICATION: histones undergo chemical modifications to help
determine chromatin configuration in a region.
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b. The N-terminus of the histone protrudes outward from the nucleosome and
is referred to as a histone tail.
c. Modifying enzymes catalyze the addition or removal of chemical groups.
27.
HISTONES AND EUKARYOTIC GENE EXPRESSION
a. Histone tails subject to post-translational modifications such as acetylation,
methylation, phosphorylation and ubiquitylation.
b. HISTONE CODE: the particular combinations of post-transcriptional
modifications found in different regions of chromatin.
28.
a.
b.
c.
d.
HISTONE ACETYLATION
Acetylation of histone tails: promotes loose chromatin structure that permits
transcription.
In histone acetylation, acetyl groups (–COCH3) are attached to lysines in
histone tails (a reversible action).
This neutralizes their positive charges and the histone tails no longer bind to
adjacent nucleosomes.
Acetylation of histone tails promotes loose chromatin structure that permits
transcription.
29.
HISTONES AND CHROMATIN REMODELING – Methylation of tails
a. The addition of methyl groups – CH3 to histone tails (methylation) can
promote condensation of the chromatin and reduced transcription.
30.
METHYLATION AND EUKARYOTIC GENE EXPRESSION
a. DNA Methylation: the presence of methyl groups on the DNA bases (usually
cytosine) of plants, animals and fungi.
b. DNA methylation plays a direct role in gene expression and is generally
associated with the silencing of DNA.
31.
a.
b.
c.
d.
EPIGENETIC INHERITANCE
EPIGENETICS: Heritable modifications in gene function not due to changes
in the base sequence of the DNA of the organism.
Can be passed on to future generations of cells.
Whereas mutations in DNA are permanent changes; modifications of
chromatin can be reversed.
DNA methylation patterns are typically erased and reestablished during
gamete formation.
32.
EUKARYOTIC GENE EXPRESSION AND GENOMIC IMPRINTING
a. IMPRINTING: a normal process.
b. Occurs in the germline of one parent, but not the other.
c. Mechanism of imprinting involves differential methylation of DNA during
germ-line differentiation.
33.
GENOMIC IMPRINTING
a. Sex specific imprinted alleles express only maternal or paternal copy.
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b. Germ cells erase imprint from your parents.
34.
GENOMIC IMPRINTING
a. 3. Imprinting established in gametes in a sex specific manner.
b. 4. After fertilization, imprint controls gene expression is somatic tissues of
embryo.
c. 5. Persists into adulthood.
35.
a.
b.
c.
d.
e.
f.
36.
a.
b.
c.
d.
e.
f.
g.
37.
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PRADER-WILLI SYNDROME
Dysmorphic syndrome
Mental Retardation
Obesity, excessive, indiscriminate eating habits (McDonalds, locks)
Small hands and feet.
Short stature hypogonadism.
~70% of cases: deletion of 15q11-q13 on chromosome inherited from
father.
ANGELMAN SYNDROME
Unusual facial appearance.
Severe mental retardation.
Short stature, spasticity, and seizures.
~70% of cases, deletion of 15q11-q13 on chromosome inherited from
mother.
Parental origin of genetic material can have a profound effect on clinical
expression of a defect.
Deletion of this region of chromosome 15 during male meiosis gives rise to
children with Prader-Willi because they are missing genes that are active
only on the paternal 15 and vice a versa.
Paternal origin of genetic material has a PROFOUND effect on the clinical
expression of a defect.
UNIPARENTAL DISOMY
a. CAN OCCUR DUE TO A TRISOMIC RESCUE
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