Gene Regulation

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Gene Regulation

• Wednesday, August 6

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Figure 19.0 Chromatin in a developing salamander
ovum
Organization of the Eukaryotic Genome

• Humans have a ton of DNA
• 35,000 genes
• 2x108 nt pairs/ chromosome
• DNA must be highly organized
• Associates with proteins to form chromatin
• Chromatin is ordered into high level structures

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Figure 19.1 Levels of chromatin packing

• Nucleosomes
• DNA wrapped around a protein core
• 2 of each histone protein (H2a, H2b, H3, H4)
• H1 histone binds between nucleosomes
• Leave DNA during replication
• Remain during transcription changes shape and
  position to allow polymerases access to the DNA

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Figure 19.1 Levels of chromatin packing

• 30nm chromatin fiber
• The string of nucleosomes coils to form a fiber
  with a diameter of 30nm
• Looped domains
• The 30nm fiber is looped and attached to a
  protein scaffold

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Figure 19.1 Levels of chromatin packing

• Metaphase chromosome
• Maximally compacted chromosome
• Selective condensation organize regions of
  transcription
• Heterochromatin highly condensed
• Untranscribed DNA
• Euchromatin less condensed
• Actively transcribed DNA

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Figure 19.x1a Chromatin
Figure 19.x1b Chromatin, detail
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Noncoding DNA in Eukaryotes

• Genes make up only a tiny portion of the genome
• Most of the genome consists of noncoding DNA
  (97)
• Regulatory elements
• promoter
• Introns
• noncoding segments of genes
• Repetitive DNA
• nucleotide sequences present in many copies in a
  genome

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Table 19.1 Types of Repetitive DNA
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Gene Families

• Multigene family
• Collection of identical or very similar genes
• Identical genes
• Clustered tandemly
• Eg genes for RNA products
• Allows for the synthesis of many copies of rRNA
• Nonidentical genes
• Clustered or dispersed
• Eg hemoglobin subunits (a and ß)
• Different versions of subunits expressed at
  different times in development
• Evolved by duplication of ancestral genes

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Figure 19.2 Part of a family of identical genes
for ribosomal RNA
3 of 100s of copies of rRNA transcription
units Each unit being transcribed by 100 RNA
polymerases Transcriptional units are separated
by spacers of nontranscribed DNA RNA transcripts
are cleaved into 3 rRNA molecules
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Gene Families

• Multigene family
• Collection of identical or very similar genes
• Identical genes
• Clustered tandemly
• Eg genes for RNA products
• Allows for the synthesis of many copies of rRNA
• Nonidentical genes
• Clustered or dispersed
• Eg hemoglobin subunits (a and ß)
• Different versions of subunits expressed at
  different times in development
• Evolved by duplication of ancestral genes

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Figure 19.3 The evolution of human ?-globin and
?-globin gene families
Hemoglobin is 4 polypeptide subunits 2a and
2ß
Pseudogenes are nonfunctional nucleotide
sequences very similar to the functional genes
In each family, genes are arranged in order of
their expression during development
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Genome Alteration

• DNA of somatic cells can be alter in a specific
  way during its lifetime
• These changes are not in the germline cells, so
  they are not passed on to offspring
• May have major effects on gene expression within
  particular cells and tissues
• Gene amplification
• Selective replication of certain genes
• Example in a developing ovum, additional copies
  of rRNA genes exist in the nucleus as tiny DNA
  circles separate from the chromosomes increases
  expression of rRNA genes
• Selective Gene Loss
• Genes are selectively lost in certain tissues
• Rearrangements
• Shuffling of substantial stretches of DNA

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Transposons

• Stretches of DNA that can move from one location
  to another within the genome mobile genetic
  elements
• Insert within a gene
• interrupts gene and prevents its normal
  functioning
• Inserts within the promoter of a gene
• cause increased or decreased transcription of
  that gene
• Inserts downstream of an active promoter
• Actives transcription of transposon gene
• Make up 10 of the human genome mostly
  retrotransponsons
• DNA ? RNA transcript ? DNA
• via reverse transcriptase, which is encoded in
  the transposon itself

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Figure 19.4 The effect of a transposon on flower
color
A transposon inserted into the gene determining
purple flower color, disrupting it and causing
the portions of the flower with this interruption
to be white.
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Transposons

• Stretches of DNA that can move from one location
  to another within the genome mobile genetic
  elements
• Insert within a gene
• interrupts gene and prevents its normal
  functioning
• Inserts within the promoter of a gene
• cause increased or decreased transcription of
  that gene
• Inserts downstream of an active promoter
• Actives transcription of transposon gene
• Make up 10 of the human genome mostly
  retrotransponsons
• DNA ? RNA transcript ? DNA
• via reverse transcriptase, which is encoded in
  the transposon itself

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Figure 19.5 Retrotransposon movement
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Immunoglobulin Genes a special case of
rearrangement

• Immunoglobulin genes code for antibodies (Abs)
  made by B cells of the immune system
• Each antibody is specific for recognizing a
  specific pathogen
• Abs consist of 4 polypeptides joined by disulfide
  bridges that contain a constant (C) region and a
  variable (V) region
• Set of genes undergo permanent rearrangement of
  DNA segments during development cell
  differentiation
• Functional Abs are pieced together from segments
  of DNA regions that are physically separated in
  the genome of an embryonic cell

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Antibody Diversity

• Variable regions of Abs give a particular Ab its
  unique ability to bind specific foreign molecules
• There are hundreds of segments of variable
  regions in the genome
• Variable and constant regions are joined and the
  intervening DNA deleted
• Combining different variable and constnat regions
  creates enormous variety of different
  polypeptides
• Further diversity results from different
  combinations of these polypeptides forming the Ab
  molecule

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Figure 19.6 DNA rearrangement in the maturation
of an Ab gene
100s
10s
1 or more
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Control of Gene Expression General Mechanisms

• Genes are turned on and off in response to
  extracellular and intracellular signals
• Gene expression is controlled on a long-term
  basis for cell differentiation
• A typical human cell expresses 3-5 of its genes
  at any given time
• Regulation occurs most commonly at the
  transcriptional level by DNA-binding proteins
  that interact with other proteins and signals
• Other stages in the pathway from gene to protein
  provide opportunities for control

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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells
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Chromatin Remodeling

• Chromatin can pack DNA in different ways,
  regulating access to it for certain
  transcriptional proteins
• Heterochromatin is highly condensed
• Inactive genes not expressed
• Euchromatin is less condensed
• Active genes expressed
• Chemical modification of chromatin can alter its
  structure allowing more or less access to the
  DNA, thereby regulating transcription

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Chromatin Modifications

• DNA Methylation
• Attachment of methyl groups (-CH3) to DNA bases
• Methylating genes can turn them off
• Demethylating genes can turn them on
• Methylation patterns are passed on during
  replication
• Histone Acetylation
• Attachment of acetyl groups (-COCH3) to certain
  amino acids of histone proteins
• Acetylation alters proteins to loosen their hold
  on DNA
• Deacetylation allows histones to grip DNA more
  tightly

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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells
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Transcription Initiation

• Once the DNA is unpacked and accessible to
  transcription proteins, specific control proteins
  fine-tune gene expression
• Regulated at initiation by interaction of
  regulatory proteins (transcription factors) with
  DNA sequences (control elements)
• Control elements are segments of noncoding DNA
  that regulate transcription of a gene by binding
  transcription factors

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Figure 19.8 A eukaryotic gene and its transcript
Control elements 1. Promoter assembles the
transcription initiation complex 2. Proximal
control elements located near the promoter 3.
Distal control elements (enhancers) located
upstream or downstream of the gene
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Transcription Factors

• Essential for transcription of all protein
  encoding genes
• Initiation complex at promoter
• Factor binds TATA box
• Other factors bind proteins
• RNA pol binds start site
• Activators transcription factors that bind to
  enhancers (close or far away) and activate
  transcription
• Repressors transcription factors that bind to
  silencers and repress transcription

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Figure 19.9 A model for enhancer action
Activator proteins bind enhancers
DNA bends to bring the activators close to the
promoter
Activators bind transcription factors and help
them form an active transcription complex on the
promoter
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Structure of Transcription Factors

• DNA-binding domains (motifs) region that binds
  DNA
• Helix-turn-helix
• Zinc finger
• Leucine zipper
• Protein-binding domain region that binds other
  transcription factors
• A particular combination of control elements
  associated with a gene allow for unique
  regulation of individual genes

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Figure 19.10 Three of the major types of
DNA-binding domains in transcription factors
Two a helices with regularly spaces leucines
wrapped around each other
Regulatory proteins
a helix and ß sheets held together by a zinc atom
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Coordinately Controlled Genes in Prokaryotes

• Metabolic pathways require enzymes for each step
  of the sequential pathway
• Bacteria can regulate the gene expression of all
  these proteins simultaneously
• Affect activity of initial enzymes
• Affect expression of all enzymes

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Figure 18.19 Regulation of a metabolic pathway
Negative Feedback Inhibition
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Coordinated Control

• Negative Feedback
• Repressible Operons
• Trp operon
• Inducible Operons
• Lac operon
• Positive Feedback
• cAMP receptor protein

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Repressible Operon

• Enzymes involved in one pathways are clustered
  together under the regulation of a single
  promoter (transcriptional unit)
• One long mRNA is transcribed and translated into
  separate polypeptides
• Grouping of functionally related genes allows
  them to be controlled as a unit by an operator
• Positioned between promoter and first enzyme
• Controls access of RNA pol to genes
• Example the trp operon (synthesis of the amino
  acid tryptophan)

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Figure 18.20a The trp operon regulated
synthesis of repressible enzymes
A regulatory gene codes for a repressor that
switches off the gene. Repressor (trpR) binds
to the operator and blocks access to the RNA pol.
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Figure 18.20b The trp operon regulated
synthesis of repressible enzymes
As tryptophan accumulates, it inhibits its own
production by activating the repressor protein
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Figure 18.20b The trp operon regulated
synthesis of repressible enzymes (Layer 2)
The repressor switches the operon off by binding
and blocking access to the promoter
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Inducible Operons

• The lac operon
• The 3 enzymes necessary to metabolize lactose are
  clustered in an operon
• lacZ B-galactosidase
• Hydrolyzes lactose to glucose and galactose
• lacY permease
• Transports lactose into the cell
• lacA transacetylase
• Lac Repressor lacI

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Figure 18.21a The lac operon regulated
synthesis of inducible enzymes
The lac repressor is synthesized in its active
form
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Figure 18.21b The lac operon regulated
synthesis of inducible enzymes
Allolactose binds and inactivates the repressor
When lactose is present, it induces the
transcription of enzymes for its synthesis
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Positive Regulation

• Lactose is only broken down in the absence of
  glucose
• cAMP (cyclic AMP) accumulates when glucose is
  scare
• cAMP binds CRP (cAMP receptor protein) and
  activates it
• CRP stimulates gene expression from the lac operon

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Figure 18.22a Positive control cAMP receptor
protein
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Figure 18.22b Positive control cAMP receptor
protein
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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells
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Figure 19.11 Alternative RNA splicing

• Different mRNA molecules are produced from the
  same transcript depending on which RNA segments
  are treated as exons and introns
• More than one type of polypeptide can arise from
  a single gene
• Regulatory proteins determine exon-intron choice

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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells
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Other Stages of Gene Control

• mRNA degradation
• The length of time a mRNA molecules exists in the
  cytoplasm is directly related to the number of
  proteins that will be synthesized from the mRNA
  (eg RBC gene hemoglobin)
• Cap and polyA tail removal begin degradation
• Translational control
• Regulatory proteins can block the initiation of
  translation by preventing the ribosome from
  attaching (eg mRNA in ovum not translated until
  specific stages of development)
• Post-translational control
• Many proteins are processed (eg proinsulin
  cleaved to functional insulin)
• Many proteins are chemically modified (addition
  of sugar or lipid molecules)
• Many proteins are transported to specific
  locations

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Figure 19.12 Degradation of a protein by a
proteasome

• The lifespan of a protein is regulated by its
  selective degradation (eg cyclins)
• Cells mark a protein for destruction by tagging
  it with a small ubiquitin molecule
• This targets the proteins to the proteosome which
  degrades the tagged protein

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Figure 19-12x Proteasomes
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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells
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Cancer

• Cells escape from the normal control mechanisms
  limiting their life
• Mutations in genes regulating cell growth and
  division the cell cycle
• Spontaneous mutation
• Carcinogens cancer inducing chemicals
• Oncogenes cancer causing genes in viruses
• Humans have normal versions of these genes called
  proto-oncogenes
• Tumor suppressor genes normally prevent
  uncontrolled growth

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Figure 19.13 Genetic changes that can turn
proto-oncogenes into oncogenes leading to an
increase in the amount or activity of the protein
product