XII. Gene Regulation

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XII. Gene Regulation
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- 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.
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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.
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And changes occur through time, creating
developmental changes. We will look at how gene
expression is regulated in these cases.
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• - 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.

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• - 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.

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• - 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.

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• XII. Gene Regulation
• A. The lac Operon in E. coli

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• 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?
  )

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• 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 gt 1 protein involved in a particular
metabolic pathway.
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• 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 gt 1 protein involved in a particular
metabolic pathway.
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• XII. Gene Regulation
• A. The lac Operon in E. coli

Lac Y - permease increases absorption of lactose
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• 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
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• 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.
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• XII. Gene Regulation
• 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
Operator
Repressor
RNA Poly
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• XII. Gene Regulation
• 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
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The binding of lactose changes the shape of the
repressor (allosteric reaction) and it cant bind
to the operator.

• XII. Gene Regulation
• 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
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• XII. Gene Regulation
• The lac Operon in E. coli
• Mutant analyses confirmed these results

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• XII. Gene Regulation
• The lac Operon in E. coli
• Mutant analyses confirmed these results

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• XII. Gene Regulation
• The lac Operon in E. coli
• Mutant analyses confirmed these results

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• XII. Gene Regulation
• 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).
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• XII. Gene Regulation
• 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 its glucose the cell needs.
  If glucose is present, there is no need to break
  lactose down to get it. BUT HOW?

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• XII. Gene Regulation
• 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 its 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.

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• XII. Gene Regulation
• 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
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• XII. Gene Regulation
• 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
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• XII. Gene Regulation
• 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
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CAP
REPRESSOR
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• XII. Gene Regulation
• 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
  nutrientand then by the presence of lactose,
  which could be metabolized to produce glucose if
  necessary.

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• XII. Gene Regulation
• The lac Operon in E. coli
• B. The trp Operon in E. coli

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• XII. Gene Regulation
• 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).

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B. The trp Operon in E. coli
If trp is absent, the repressor cant bind to the
operator transcription proceeds..
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B. The trp Operon in E. coli
If trp is present, it binds to the repressor,
changing the repressors shape so that it can now
bind to the operator and inhibit RNA poly binding.
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B. The trp Operon in E. coli
Secondary Regulation
Actually, when trp is present,
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B. The trp Operon in E. coli
Secondary Regulation
ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE
BITUP TO THE REGION CALLED THE ATTENUATOR
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B. The trp Operon in E. coli
Secondary Regulation
ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE
BITUP TO THE REGION CALLED THE ATTENUATOR
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B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the m-RNA the 3-4
loop causes termination of transcription.
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B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the m-RNA 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.
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B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the m-RNA 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
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B. The trp Operon in E. coli
Secondary Regulation
Two hairpin loops can form in the m-RNA 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
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• XII. Gene Regulation
• The lac Operon in E. coli
• B. The trp Operon in E. coli
• C. Regulation in Eukaryotes

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• XII. Gene Regulation
• 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.

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• XII. Gene Regulation
• 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.
• 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.

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• C. Regulation in Eukaryotes
• Histone Regulation
• - Three ways to reveal DNA
• chromatin remodeling

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• C. Regulation in Eukaryotes
• Histone Regulation
• - Three ways to reveal DNA
• chromatin remodeling
• Methylation
• - highly repetitive sequences
• - imprinted genes
• - Barr bodies

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• C. Regulation in Eukaryotes
• Histone Regulation
• - Three ways to reveal DNA
• chromatin remodeling
• 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.

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• C. Regulation in Eukaryotes
• Histone Regulation
• Methylation
• Promoters
• - Several consensus sequences (TATA, CAAT,
  GGGCGG) appear in combination in nearly all
  promoters and are required for basal levels of
  transcription

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• C. Regulation in Eukaryotes
• Histone Regulation
• Methylation
• Promoters
• 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

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• C. Regulation in Eukaryotes
• Histone Regulation
• Methylation
• Promoters
• 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.

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• C. Regulation in Eukaryotes
• Histone Regulation
• Methylation
• Promoters
• 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?

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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.
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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• Transcription Factors
• - These are the proteins that bind to DNA and
  influence transcription. They have binding
  domains that bind DNA in particular ways.

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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• 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 TFs contain specific
sequences of AAs called a homeodomain. This is
encoded by a 180 bp region in its gene called a
homeobox. These homeotic genes/proteins are
conserved across all eukaryotes and are critical
to basic animal development.
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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• 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 AAs. The sequence between forms
• A loop or finger, and the specific AA sequence
• Binds specific DNA sequences

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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• Transcription Factors

We didnt really know what they did in vivo.
Biochemists have linked other proteins to them,
however, making Zinc-finger nucleases that cut
DNA at specific sequences.
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(No Transcript)
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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• 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 (gt 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.

Science Daily
READ THIS
Najafabadi et al. 2015
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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• Transcription Factors
• - These are the proteins that bind to DNA and
  influence transcription. They have binding
  domains that bind DNA in particular ways.
• bZIPbasic leucine zipper leucine AAs in
• Different chains dimerize and the leucines zip
• The other alpha-helices bind specific DNA
  sequences

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• C. Regulation in Eukaryotes
• Enhancers/Silencers
• 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 TFs have other binding sites for
• Proteins (like basal transcription factors) or
• Other chemicals (like hormones)

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• C. Regulation in Eukaryotes
• Transcription Factors
• 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
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• C. Regulation in Eukaryotes
• 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.
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• C. Regulation in Eukaryotes
• Controlling m-RNA stability
• RNA Silencing
• - Short pieces of RNA can bind to DNA in the
  nucleus or m-RNA in the cytoplasm and regulate
  gene expression.

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• C. Regulation in Eukaryotes
• Controlling m-RNA stability
• 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.

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• C. Regulation in Eukaryotes
• Controlling m-RNA stability
• 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.

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• C. Regulation in Eukaryotes
• Controlling m-RNA stability
• 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.

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• C. Regulation in Eukaryotes
• Controlling m-RNA stability
• RNA Silencing/Interference
• OR!
• The ds-RNA gets complexed with RNA-induced
  initiation of transcription silencing complex
  (RITS). These denature the RNA, creating ss-RNA
  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).

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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 Transcriptio
n Transcript Processing Translation Post-transl
ational Modification
Variation in patterns of regulation lead to
differences in expression between cells, and cell
specialization.