Download MCB Lecture 3 – Gene Regulation

MCB Lecture 3 – Gene Regulation
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What is an inducible gene?
o An inducible gene is normally off, but can be turned on.
What is a repressible gene?
o A repressible gene is always on, but can be turned off.
What are constitutive genes?
o Constitutive genes are those that stay more or less constant because
they are the housekeeping genes of the cell.
What are three ways to measure gene expression?
o Northern Blot
o RT-PCR
o DNA Microarray
What is the main determinant for expression of housekeeping genes?
o The deviations from the consensus sequence reduces promoter
function, leading to differences in mRNA product
What are specificity factors?
o They alter the specificity of RNA Polymerase for promoter in
transcription
What is the specificity factor in Bacteria and Eukaryotes?
o Bacteria: Sigma Factor
o Eukaryotes: TBP (Tata Binding Protein)
What is a repressor? What type of regulation is it?
o A Repressor impedes access of RNA Polymerase to the promoter. It is
negative regulation.
What is an activator? What type of regulation is it?
o An Activator enhances RNA Polymerase – Promoter interaction. It is
positive regulation.
What two signal molecules act on a repressor? What do they do?
o Co-repressors: bind to Repressor to enhance the binding to the
operator so that RNA Polymerase cannot bind to the promoter.
o Inducers: Inducers bind to the repressor to prevent it from binding to
the operator, so that RNA Polymerase can bind.
What two signal molecules act on an activator? What do they do?
o Co-Activators: Binds to the activator to enhance the binding of it so
that it can help RNA Polymerase bind to the strand.
o Repressors: Binds to the activator so that the activator cannot bind to
the strand, preventing RNA Polymerase from binding.
Do you get transcription when you have:
o Co-Activator + Activator? YES
o Inducer + Repressor? YES
o Co-Repressor + Repressor? NO
o Repressor + Activator? NO
What is an operon? Where are they found?
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o Operons are in Prokaryotes only. They are clusters of genes involved
in the same biochemical pathway.
What is the name of the sequence in the operon where the repressor binds?
o Operator
What is the product of an operon?
o A Polycistronic mRNA
LAC OPERON:
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What type of organism utilizes the Lac Operon?
o E. Coli
What type of energy source does it prefer?
o Glucose
Why does it use Lactose as a backup energy source?
o Because Lactose is made from Galactose and Glucose. Lactose can be
broken down to get Glucose (which they prefer)
What does the enzyme Galactoside Permease do?
o Allows Lactose into the cell.
What does B-Galactosidase do?
o It breaks down Lactose into Allolactoe and then Galactose/Glucose
Where are the lacZ, lacY, and lacI genes located?
o lacZ and lacY are in the operon. lacI is a repressor gene located
somewhere else in the genome
What is the role of Allolactose?
o Allolactose acts as an inducer. It binds to the repressor, so that the
repressor is removed from the operon and transcription of the gene
can occur by RNA Polymerase
cAMP Responsive Protein (CRP or CAP) does what for the Lac Operon?
o CRP (CAP) is an activator. cAMP levels are lowered by the presence of
Glucose. However, when little Glucose is present, the cAMP levels go
up. cAMP acts as a co-activator for CRP (CAP), which then binds to the
Lac Operon to activate it.
Under these conditions, will the Lac Operon be transcribed? What is present
on the strand for each one (activators, repressors, etc.)?
o + Glucose, +Lactose = NO
 Present on Strand: Nothing
o +Glucose, -Lactose = NO
 Present on Strand: No CRP (because high glucose lowers cAMP
levels). Because there is no lactose to bind to the repressor (as
an inducer), the repressor IS on the strand.
 Repressor: YES; Activator: NO
o –Glucose, -Lactose = NO
 Present on Strand: There is CRP on the strand because low
glucose levels means high levels of cAMP. The cAMP (as a co-
activator) binds to CRP, which allows it to bind to the strand.
However, the lack of Lactose means that no Allolactose can
bind to the repressor (as an inducer), so the repressor is also
on the strand.
 Repressor: YES; Activator: YES
o –Glucose, +Lactose = YES
 Present on Strand: There is CRP on the strand because the low
glucose levels means high levels of cAMP. The cAMP (as a coactivator) binds to the CRP, which allows it to bind to the
strand. The presence of Lactose allows Allolactose to bind to
the repressor (as an inducer). This removes the repressor
from the strand and allows transcription to occur.
Tryptophan Operon:
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What is the Trp Operon?
o It consists of a promoter region and 5 genes involved in biosynthesis
of tryptophan.
Which region does the repressor protein recognize?
o The Promoter Region - Operator
What is the function of Trp?
o It acts as a co-repressor to the repressor. Two Trp molecules must be
bound to effectively bind to the operator
Where is the repressor transcribed?
o Elsewhere in the E. Coli genome. It is constitutively expressed
(always present)
What happens when there is high Trp?
o Trp acts as a co-repressor and binds to the repressor, which can then
bind to the operator and prevent transcription
By how much can the Trp repressor reduce expression?
o Factor of 70
By how much can Attenuation decrease expression of Trp operon?
o Factor of 10
How much, collectively, can attenuation and Trp Repressor reduce
expression of the Trp Operon?
o 700 Fold Collectively
What is attenuation?
o A mode of transcriptional regulation of operons in prokaryotes by
prematurely terminating transcription.
Where does attenuation happen?
o In the Open Reading Frame, which is upstream of the structural genes
(14 amino acids)
What is a leader peptide?
o The sequence of mRNA in the ORF that codes for two Trp’s.
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What happens in attenuation when there are low levels of Trp?
o When there are low levels of Trp, the Leader Peptide stalls at the code
for the two Trp’s. This allows the 2:3 hairpin binding, and attenuation
is prevented (so the strand can be transcribed and Trp can be made)
What happens in attenuation when there are high levels of Trp?
o When there are high levels of Trp, the Leader Peptide is able to
synthesize the protein quickly because Trp’s are available. Therefore,
it moves through quickly and does not give the 2:3 time to hairpin.
Because of this, the 3:4 hairpin forms instead, which prevents
transcription any further.
What type of transcriptional regulation (that was previously discussed) is
this?
o Rho-Independent Termination (hairpins)
Regulation of Eukaryotic Gene Transcription
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How does the structure of Heterochromatin regulate transcription?
o It is too condensed for transcription to happen, so you get no gene
expression.
How does the structure of Euchromatin regulate transcription?
o Euchromatin is sections of the DNA that are unwound and allow
transcription to occur. (Only particular regions)
What are Hypersensitive Sites?
o Regions in chromosomes that are devoid of nucleosomes and allow
for transcriptions factors to bind (so genes can be expressed)
o They are upstream of the transcription site
What is used as a lab tool to degrade DNA (for Hypersensitive Sites).
o DNase
What are Histone Acetyl Transferases (HATs)?
o They covalently modify the core histones of nucleosomes (specifically
H3 and H4) by adding acetyl groups and making histones less positive.
When the histones are less positive, they have a weaker bond to the
negatively charged DNA.
o They transfer this to Lysine and Arginine!!
What are Histone Deacetylases?
o They covalently modify the core histones of nucleosomes (specifically
H3 and H4) by removing acetyl groups and making the histones more
positive. When the histones are more positive, they have a stronger
bond to the negatively charged DNA.
What are SWI/SNF Complexes?
o They are remodeling proteins that use ATP to remodel nucleosomes
by repositioning them or removing them
What is Rubinstein-Taybi Syndrome?
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o Caused by a deficiency of Histone Acetyl Transferase, due to mutation
in CBP
 When Histone Acetyl Transferase is not working properly, it is
unable to weaken the bond between DNA and Histone
Proteins. This means that those regions cannot be expressed.
o Patient presents with: Growth deficiency, microcephaly, broad
thumbs, big toes, and mental retardation.
What is an enhancer?
o It is an element on the DNA where transcription factors can bind to
increase the activity of transcription
o Can be close (acting in CIS) or come from far away (acting in TRANS)
What is an insulator?
o They limit the RANGE of enhancers by binding to the DNA and
forming looped domains, which act as a physical block.
o They decrease the activity of transcription
What is a Barrier Sequence?
o It prevents spreading of the heterochromatic state of DNA
What is Deletion Mapping?
o It is used to find regulatory sequences (positive and negative) by
deleting particular regions of DNA and looking at the effect on
Promoter Activity
In Deletion Mapping, what type of deletion causes an increase in Promoter
Activity?
o A deletion of a repressor
In Deletion Mapping, what type of deletion causes a decrease in Promoter
Activity?
o A deletion of a activator
What is needed for RNA Pol II to bind to a promoter?
o General Transcription Factors:
 TFIID, TFIIB, TFIIF, TFIIE, TFIIH
o Trans-activators (which bind to enhancers)
o Co-activators
o Mediator Co-activators
What two types of activity does TFIID have?
o General Transcription Factor Activity
o Co-Activator Activity
What is the mechanism by which a Steroid Hormone binds to a Nuclear
Hormone Receptor for Gene Activation?
o 1. Hormone enters the cell by diffusion
o 2. Binds to the hormone receptor in the cytoplasm
o 3. Receptor translocates to the nucleus (usually a dimer)
o 4. The receptor acts as a transcription factor by binding to the DNA
Binding Domain (DBD) in the Major Groove
o What is the name of the promoter element that the Receptor binds to?
 Hormone Response Element (HRE) – Palindromic Repeat
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o What is an example of a Steroid Hormone that does this?
 Glucocorticoid
What is the mechanism for transcriptional regulation done by
Glucocorticoids?
o 1. The Glucocorticoid Receptor (R) is normally bound by the HSP 90
and is inactive.
o 2. Binding of Glucocorticoid to the Glucocorticoid Receptor releases it
from HSP 90, which induces the dimerization of R (RR)
o RR moves to the nucleus (as a Dimeric Receptor DBD)
o RR activates HRE element through chromatin remodeling coactivators (HATs)
o ** When the glucocorticoid is available to bind to the receptor,
different transcription factors can lead to different effects of gene
expression based on where the receptor binds.
How does Tamoxifen treat breast cancer?
o It is a competitive antagonist of the estrogen receptor.
o It binds to the estrogen receptor as a CO-REPRESSOR
o This blocks cell division
Protein Phosphorylation and Gene Activation
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How does Epinephrine act in Gene Activation?
o 1. Epinephrine activates G-Protein Coupled Receptors
o 2. Activated G-Protein binds to and activates Adenylyl Cyclase
o 3. cAMP level increases
o 4. cAMP binds to Regulatory Subunit of Protein Kinase A (PKA)
o Released PKA Catalytic Subunits move to the nucleus
o 5. PKA phosphorylates the cAMP-Response Element Binding Protein
(CREB) in the nucleus
o 6. CREB binds to cAMP Response Element (CRE) in the Promoters of
Target Genes
o 7. CREB Binding Protein (CBP) binds as a co-activator
o 8. Histone Acetylation and Chromatin Remodeling facilitate
Transcription.
Co-Repressors:
o What type of activity do co-repressors have?
 Histone Deacetylase (HDAC) Activity
 This removes acetyl groups so that the Histones tightly
bind to the DNA and it cannot be transcribed
o Other ways co-repressors work:
 1. Histone Methyl Transferases
 2. H3-K9 Methylation
 Lysine Residue #9 of Histone H3 Methylation
Decreasing Transcription: Methylation and De-acetylation
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Where in Eukaryotic DNA can methyl groups be added?
o It is limited to Cytosine bases in CpG dinucleotides
What is the enzyme called that adds methyl groups?
o DNA Methylase
Does methylating DNA silent a gene or activate a gene?
o Silences it
Discuss the MeCP example for Histone Methylation.
o 1. MeCP Proteins bind to Methylated CpG nucleotides
o 2. MeCP 2 recruits HDAC Activitiy (removed acetyl groups)
 This induces chromatin remodeling
o 3. MeCP2 aids in methylation of H3K9 (Lys9 on Histone H3)
o 4. HP1 (Heterochromatin Protein 1) binds to Methylated H3K9 and to
itself, condensing the chromatin
DNA/Protein Interactions
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DNA-Protein interactions can depend on two types of bonds – what are they?
o Hydrogen Bonding
o Hydrophobic Interactions
What are the amino acids involved in DNA Binding?
o N – Asparagine
o Q – Glutamine
o E – Glutamate
o R – Arginine
o K – Lysine
What is the DNA target site usually?
o A Palindrome
What is a Helix-turn-Helix Motif?
o A DNA/Protein Interaction that can be found in Bacteria and Humans
o It is a helical region interacting with the DNA
o Rich in NQERK
 N – Asparagine
 Q – Glutamine
 E – Glutamate
 R – Arginine
 K – Lysine
What is an example of the Helix-Turn-Helix in bacteria?
o The Lac Repressor
How does the Helix-turn-Helix function in Humans?
o It is found in the homeodomain on transcription factors
o It binds to the homeobox on DNA
What is an example of Helix-turn-Helix in Humans?
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o HOXD13
What causes Synpolydactyly?
o A mutation on the HOXD13 gene.
o The homeobox is mutated (DNA) so transcription factors can not bind
What is a homeodomain?
o A region on protein that binds to complementary DNA
What is a homeobox?
o A region on the DNA in which the protein binds
What are Zinc Fingers?
o A DNA/Protein Interaction in Humans
o Zinc Fingers do not directly interact with DNA. They interact with
Transcription Factors
o Consists of Zinc 2+ions held by 4 Cysteines OR 2 Cysteines and 2
Histines
o One Zinc Fingers yields a weak interaction with DNA, but multiple
Zinc Fingers increase the strength
What is the name of a transcription factor that uses zinc fingers?
o TFIIA
What is an HMG-Box Motif?
o High Mobility Group-Box Motif
o Consists of three alpha-helical (protein) regions that interact with
DNA
o The DNA bends, allowing for interaction with other transcription
factors on nearby sites.
What is an example of an HMG-Box Motif?
o SRY Gene – Responsible for male sexual differentiation and contains
HMG Box.
Protein-Protein Interactions
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What are Leucine Zippers?
o Leucine-Rich Regions on Transcription Factors that allow for the
binding of one transcription factor to another, which increases
transcription
o Have bZIP domains that interact with DNA
What is a Helix-Loop-Helix?
o Involved in protein-dimer formation
What is an example of a Helix-loop-Helix?
o MyoD transcription factors use Helix-Loop-Helix
Regulating Translation
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How does negative feedback affect translation in ribosomal protein operons?
o The mRNA synthesizes Ribosomal Proteins.
o The Ribosomal Protein (r-protein) then binds to its mRNA and
prevents further translation of the mRNA
What is an example of how protein kinases affect the regulation of
translation?
o Globin Synthesis in Reticulocytes
What is globin synthesis dependent on?
o The phosphorylation of eIF2.
Explain the mechanism of Protein Kinases in the Initiation of Translation.
o eIF2 is stable when it is dephosphorylated
o eIF2 is unstable with phosphorylated by KINASE
o Heme inhibits Kinase
o When Iron is present, Heme is Present
o When Iron is present, Kinase is inhibited and eIF2 is stable and active
o When Iron is not present, there will be no heme. The Kinase can
phosphorylate eIF2 and deactivate it (it gets degraded)
What is an Iron Responsive Element (IRE)
o It is present in mRNA and it regulates the ferritin and transferrin
receptor
What is Ferritin?
o It is an iron-storage protein that gets translated when there is excess
iron
What is Transferrin?
o It is an iron-uptake protein that gets translated when there is iron
deficiency
During Iron Starvation, what will the cell translate?
o Ferritin is not translated
o TRANSFERRIN IS TRANSLATED
During Excess Iron, what will the cell translate?
o FERRITIN IS TRANSLATED
 The addition of iron removes the Binding Protein from the 5’
IRE – removes the “road-block” so it can translate
o Transferrin is not translated.
 The addition of iron also removes the Binding Protein from the
3’ IRE – destabilizes the mRNA