Defects in gene regulation can alter

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Regulation of Gene Expression
Defects in gene regulation can alter the
development of an organism
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Seven processes that affect the steady-state
concentration of a protein in a cell
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Regulation of Gene Expression

• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes

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Principles of Gene Regulation
constitutive vs. regulated gene
expression housekeeping genes, gene products
that are required at all times at a more or less
constant level. e.g., in citric acid cycle
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Principles of Gene Regulation
1) RNA polymerase binds to DNA at promoters
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Principles of Gene Regulation (contd)
2) Transcription initiation is regulated by
proteins that bind to or near promoters.
Repression of a repressible gene(i.e., negative
regulation) repressors (vs. activators) bind
to operators of DNA. Repressor is regulated by an
effector, usually a small molecule or a
protein, that binds and causes a conformational
change. Activator binds to DNA sites called
enhancer to enhance the RNA polymerase
activity. (i.e., positive regulation) Induction
of an inducible gene, e.g., heat-shock genes.
Heat-shock promoters
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Principles of Gene Regulation (contd) 3) Most
prokaryotic genes are regulated in units
called operons.
Francois Jacob Jacques Monod, 1960
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Lactose metabolism in E. coli
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4) The lac operon is subject to negative
regulation repressor
tetrameric repressor
IPTG induced
uninduced
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5) Regulatory proteins have discrete DNA-binding
domains
Functional groups (pink) in DNA available for
protein binding
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5) Regulatory proteins have discrete DNA-binding
domains
e.g., specific amino acid-base pair interactions
in DNA-protein interaction
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e.g., a DNA-binding domain (3) interacts
directly with DNA at major groove
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The DNA binding sites for regulatory proteins are
often inverted repeats of a short DNA sequence
(a palindrome) at which multiple subunits
(usually two) of a regulatory protein bind
cooperatively.
inverted repeats
e.g., Lac repressor vs. operator
AATTGTACAATT TTAACATGTTAA
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• Examples of DNA-binding motifs/domains
• helix-turn-helix e.g., Lac repressor
• zinc finger e.g., Zif 268
• homeodomain e.g., Ultrabithorax (Ubx)

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Helix-turn-helix

DNA-binding domains
Lac repressor, a tetramer
allolactose-binding domains
hydrogen-bonding (red) hydrophobic interactions
(yellow)
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• zinc finger

In many eukaryotic (few prokaryotic) DNA-binding
proteins
e.g., Zif 268
30 a.a.
Zn2
(2 Cys, 2 His)
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• homeodomain
• homeobox DNA sequence encoding
• homeodomain

e.g., Ultrabithorax (Ubx)
a a helix (red) protruding into the major groove
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6) Regulatory proteins also have protein-protein
interaction domains

• Leucine zippers
• basic helix-loop-helix

interacting Leu (red)
Leucine zippers
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basic helix-loop-helix e.g., transcription factor
Max (dimeric)
A pair of interacting Leu
helix-loop-helix (red purple)
DNA-binding segment (pink)
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Regulation of Gene Expression

• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes

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The Lac Operon
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The lac Operon Is Subject to Positive
Regulation Activation by CRP (cAMP receptor
protein)
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CRP homodimer
DNA is bended
Region interacting with RNA polymerase (yellow)
cAMP (pink)
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The effect of glucose on CRP is mediated by
cAMP. Transcription occurs only at low glucose
and high lactose.
cAMP CRP are involved in the coordinated
regulation of many operons. A net of operons
with a common regulator is called regulon.
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The ara operon undergoes both positive negative
regulation by a single regulatory protein AraC.
Th end product of the arabinose metabolic
pathway, D-xylulose 5-phosphate, is an
intermediate in the pentose phosphate pathway.
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When the AraC repressor is depleted, The araC
gene is transcribed from its own promoter.
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At high glucose and low arabinose, AraC binds
and brings araO2 and araI sites together to form
a DNA loop, repressing araBAD.
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At low glucose, but arabinose is present, AraC
repressor binds arabinose and changes
conformation to become an activator. DNA loop is
opened, and AraC binds to each half-site of araI
and araO1. The proteins interact with each other,
and act in concert with CRP-cAMP to facilitate
transcription of the araBAD genes.
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Many Genes for Amino Acid Biosynthesis Are
Regulated by Transcription Attenuation e.g., the
trp operon
At high tryptophen, 1) the repressor binds its
operator, 2)
transcription of trp mRNA is attenuated.
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Trp repressor
dimeric, helix-turn-helix
bound tryptophen (red)
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Transcription attenuation in the trp operon
The trp mRNA leader (trpL) Sequence 1 encodes a
small peptide, leader peptide, containing two Trp
residues.
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Transcription attenuation in the trp operon
attenuator
At high trp
At low trp
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The Trp Operon
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Induction of the SOS Response in E. coli Requires
Destruction of Repressor Protein LexA
Operon-like regulation
Coprotease RecA is activated by DNA damage
(single stranded DNA) LexA is cleaved and
inactivated by RecA
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Synthesis of Ribosomal Proteins Is Coordinated
with rRNA Synthesis
mRNAs of some ribosomal proteins (r-protein)
r-protein acts as a translational repressor
yellow RNA pol subunits blue EFs
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Synthesis of Ribosomal Proteins Is Coordinated
with rRNA Synthesis
e.g., stringent response in E. coli, response to
amino acid starvation
uncharged tRNA binding gt stingent factor (RelA)
binding gt catalysing ppGpp synthesis gt binding to
b-subunit of pol gt rRNA synthesis reduced
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Some Genes Are Regulated by Genetic Recombination
e.g., regulation of flagellin genes in
Salmonella phase variation allows evasion of
host immune response.
repressor
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Regulation of Gene Expression

• Principles of gene regulation
• Regulation of gene expression in prokaryotes
• Regulation of gene expression in eukaryotes

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• Extraordinary complexity of gene regulation in
  eukaryotes
• Transcriptional Active Chromatin is Structurally
  Different from
• Inactive Chromatin
• hypersensitive sites (100 200 bp), DNaseI
  sensitive sequences whithin
• the 1000 bp flanking the 5 end of transcribed
  genes.
• Modifications Increase the Accessibility of DNA
• e.g., 5-methylation of cytosine of CpG
  sequences is common in
• eukaryotic DNA, active genes tend to be
  undermethylated.

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• Extraordinary complexity of gene regulation in
  eukaryotes
• Chromatin Is Remodeled by Acetylation
• and Nucleosome Displacements
• chromatin remodeling the detailed mechanisms
  for
• transcription-associated structure changes in
  chromatin.

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Extraordinary complexity of gene regulation in
eukaryotes

• Many Eukaryotic Promoters Are Positively
  Regulated
• DNA-Binding Transactivators and Coactivators
  Facilitate
• Assembly of the General Transcription Factors
• enhancer in higher eukaryotes,
• upstream activator sequences (UASs) in yeast.

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Three Classes of Proteins Are Involved in
Transcriptional Activation basal transcription
factors, DNA-binding transactivators, and
coactivators.
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A wide variety of repressors function by a range
of mechanisms
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The Genes Required for Galactose Metabolism in
Yeast Are Subject to Both Positive and Negative
Regulation
Binding of galactose to Gal3p and its interaction
with Gal80p produce a conformation change in
Gal80p that allows Gal4p to function in
transcription activation.
regulated 6 genes (table 28-3) regulatory
proteins Gal4p, Gal80p Gal3p
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Unlike bacteria, there is no operons in yeast.
Each of the GAL genes is transcribed separately.

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The GAL system is shown to illustrate the
transcription activation of a group of related
eukaryotic genes.

• The initiation complexes
• assemble stepwise
• DNA-binding transactivators
• Basal transcription factors/pol II
• Additional protein complexes
• needed to remodel the chromatin

e.g., SWI/SNF histone remodeling SAGA
histone acetylation
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Typical DNA-binding transactivators have a
DNA-binding domain and an activation
domain. e.g., Gal4p, acidic activation domain
function in activation CTF1(CCAAT-binding
transcription factor 1), proline-rich activation
domain Sp1, glutamine-rich activation
domain
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Domain-swapping experiment A chimeric protein
containing the DNA-binding domain of Sp1 and the
activation domain of CTF1 activates
transcription if a GC box is present.
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Eukaryotic Gene Expression Can Be Regulated by
Intercellular and Intracellular Signals e.g.,
steroid hormone (estrogen) receptors
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Regulation Can Occur through Phosphorylation of
Nuclear Transcription Factors e.g., The
catalytic subunit of protein kinase A, released
when cAMP levels rise, enters the nucleus
and phosphorylates a nuclear protein, the
CRE-binding protein (CREB), gtgt binding to
CREs near certain genes and acting as a
transcription factor.
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Many Eukaryotic mRNAs Are Subject to
Translational Repression e.g., translational
repressors (RNA-binding proteins) interact with
initiation factors or with the ribosome to
prevent or slow translation
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Development Is Controlled by Cascades of
Regulatory Proteins
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Maternal Genes Christiane Nusslein-Volhard
(p.1112) bicoid (bcd) gene product gradient
Two posteriors
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Segmentation genes
e.g., ftz gene product (Ftz)
Early embryo
Late embryo
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Homeotic genes
Antennapaedia ( mouse HOX 1.1)
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The discovry of structural determinants with
identifiable molecular functions is the first
step in understanding the molecular events
underlying development.
bithorax mutation