Chapter 7: Control of Gene Expression

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Chapter 7 Control of Gene Expression
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Control of Gene Expression

• Different cell types differ dramatically in
  structure and function
• same genome
• Cell differentiation depends on gene expression

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Control of Gene Expression

• Evidence for preservation of genome during cell
  differentiation

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Control of Gene Expression

• Different Cell Types Synthesize Different Sets of
  Proteins
• How many differences are there btwn any one cell
  type and another
• Many processes are common to all cells
• Some processes are cell specific
• Cell expresses 10,000-20,000 of its 30,000
  genes level of expression of almost every
  gene varies from cell to cell

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Control of gene Expression

• Cells Can Change Expression of its Genes in
  Response to External Signals
• Different cell types respond in different ways to
  same extracellular signal general feature of
  cell specialization
• Example Liver and adipocyte cells respond
  differently to glucocorticoid

Liver Cell Tyrosine aminotransferase
Adipocyte Tyrosine aminotransferase
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Control of Gene Expression
For most genes transcription control is most
important
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Control of Gene Expression

• 2 Fundamental Components to Transcriptional Gene
  Regulation
• 1. Gene Regulatory Proteins
• 2. Short Stretches of DNA of Defined Sequence

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Control of Gene Expression

• Outside of DNA Helix Read by Proteins
• GRP recognizes specific nucleotide sequence
• Information in form of
• H-bond acceptors
• H-bond donors
• Hydrophobic patches
• Bind to Major Groove

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Control of Gene Expression

• GRPs bind to major groove where patterns for ea
  of four
• base-pair arrangements are distinct

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Control of Gene Expression
Geometry of Double Helix Depends on Nucleotide
Sequence

• Some nucleotide sequences cause DNA to bend
• AAAANNN
• If repeated every 10 bp DNA appears unusually
  curved

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Control of Gene Expression
DNA must be flexible for binding of GRPs
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Control of Gene Expresion

• Short DNA Sequences Fundamental Components of
• Genetic Switches
• GRP recognition sequence generally lt 20 bp
• Thousands of such DNA sequences identified ea of
  which is recognized by different GRP

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Control of Gene Expression

• GRP DNA Interactions
• Exact fit btwn DNA and protein
• H-bonds, ionic bonds, hydrophobic
• gt 20 contacts
• Tight and specific

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Control of Gene Expression

• Major Structural Motifs of GRPs
• Helix-turn-helix
• Homeodomain
• Zinc Finger
• Leucine Zipper
• Helix-Loop-Helix

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Control of Gene Expression

• Helix-Turn-Helix
• Most common
• C-terminal helix recognition helix
• aa in recognition helix define specificity
• Structure of GRP varies outside HTH HTH
  presented in unique way

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Control of Gene Expression

• Homeodomain
• Special type of helix-turn-helix
• Conserved stretch of 60 aa
• HTH motif always surrounded by same structure-
  homeodomain
• Master regulators of development

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Control of Gene Expression

• Zinc Finger Proteins
• a helix and ß sheet
• (2) a helices

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Control of Gene Expression

• Leucine Zipper
• Clothespin
• Helices held together by short
• coiled coil region of hydrophobic
• residues often leucines

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Control of Gene Expression

• Helix-Loop-Helix
• Short a helix connected to another via loop
• Flexible loop for packing

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Control of Gene Expression

• Heterodimerization
• Enhances the repertoire of DNA binding
  specificities
• Combinatorial control

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Control of Gene Expression

• Is it possible to predict DNA sequence to which
  GRPs bind?

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Control of Gene Expression

• Gel Mobility Shift Assay to Detect GRPs
• effect of a bound protein on the migration of DNA
  in an electric field

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Control of Gene Expression

• DNA Affinity Chromatography to Purify GRPs
• Purification of GRP gt 10,000X

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Control of Gene Expression
How do we determine the sequence to which a
particular GRP binds?
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Control of Gene Expression

• Chromatin Immunoprecipitation
• Identifies sequences occupied by GRPs
• in living cells
• Used to identify direct targets of GRPs

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How Genetic Switches Work

• Tryptophan Operon

Operon a cluster of genes transcribed as a
single mRNA Operator short region of DNA in
bact. that controls transcription of an adjacent
gene
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How Genetic Switches Work

• Tryptophan Repressor a Simple On/Off Switch

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How Genetic Switches Work

• Repressor protein binds to DNA to prevent
  transcription of adjacent gene
• Activator protein that binds to DNA and
  promotes the transcription of adjacent gene

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How Genetic Switches Work

• CAP Catabolite Activator Protein
• Promotes transcription of genes that enable E.
  coli to use
• alternative carbon sources when glucose is not
  available
• glucose cAMP
• cAMP binds to CAP enabling CAP to bind to
  sequences near
• target promoters to promote transcription

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How Genetic Switches Work
More complicated genetic switches combine
positive and negative controls
Lac Operon- under the control of transcriptional
activator and transcriptional repressor
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How Genetic Switches Work

• Regulation of Transcription in Eukaryotes is More
  Complex
• GRPs can act even when positioned 1000s bp away
  from promoter
• RNA Pol II cannot initiate transcription on its
  own, requires GTFs
• Packaging of DNA in chromain

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How Genetic Switches Work

• Eucaryotic Gene Control Region
• Promoter and all regulatory sequences to which
  GRPs bind to control transcription
• gt 50,000 bp, not unusual
• Packaged in nucleosomes and higher order forms of
  chromatin

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How Genetic Switches Work

• Eucaryotic GRPs
• 5-10 of human genome
• Vary from one control region to next
• Present in sm amts, lt0.01 total protein
• Most recognize specific DNA sequences others
  assemble on other DNA bound proteins
• Allow genes to be turned on and off very
  specifically

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How Genetic Switches Work

• Eucaryotic Gene Activator Proteins Promote
  Assembly
• of RNA Polymerase and GTFs at Transcription Start

• Gene Activator Proteins have Modular Design
• DNA Binding Domain
• Activator Domain

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How Genetic Switches Work

• Mechanism of Gene Activator Proteins Varied but
  All Promote Assembly of GTFs and RNA Pol
• Interact w/ initiation complex to recruit RNA Pol
  
• Interact directly w/RNA Pol and GTFs
• Change chromatin structure around promoter

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How Genetic Switches Work

• GRPs can affect
• prescribed ordered assembly of GTFs and RNA
  Polymerase
• Recruitment of RNA Polymerase holoenzyme to
  promoter

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How Genetic Switches Work

• Gene Activator Proteins Promote Assembly of GTFs
  and RNA Pol By
• Modification of Local Chromatin Structure
  Recruiting
• histone acetyl transferases
• histone remodeling complexes

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How Genetic Switches Work
Gene Activator Proteins Work Synergistically
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How Genetic Switches Work
EX Complexity of How Gene Activator Proteins
May Ultimately Increase Transcription Rate
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How Genetic Switches Work
Eucaryotic Repressors Inhibit Transcription in
Variety of Ways
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How Genetic Switches Work

• Eucaryotic GRPs and Combinatorial Control
• Function as unit to generate complexes whose
• function depends on final assembly of all
• components
• Not designated activators or repressors
• DNA acts as nucleation site for assembly
• Can participate in gt one type of reg. complex
• Coactivators and corepressors
• enhancesome

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How Genetic Switches Work

• Eve-skipped gene is a complex multicomponent
  genetic switch in drosophilia
• Drosophilia development
• Eve expressed when embryo single giant
  multinucleated cell
• Cytoplasmmixture of GRPs distributed unevenly
  along length of embryo
• Nuclei originally identical but later express
  diff genes cuz exposed to diff GRPs

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How Genetic Switches Work

• Eve Expression
• Regulatory sequence reads conc of GRPs at ea
  position along length of embryo
• Expressed in 7 stripes 5-6 nuclei wide precisely
  positioned along anterior- posterior axis

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How Genetic Switches Work

• Regulatory Region of Eve Gene
• 20,000 bp binds gt20 proteins
• Series of regulatory modules
• Regulatory modules contain multiple reg sequences
  responsible for
• specifying a particular stripe

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How Genetic Switches Work

• Expression of Stripe 2
• Dictated by 2 gene activator proteins and 2 gene
  repressor proteins
• Transcription occurs when activators Biocoid and
  Hunchback are high
• and repressors Kruppel and Giant are low

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How Genetic Switches Work

• Combinatorial Control
• Heterodimerization of GRPs in soln
• Assembly of combos of GRPs into sm complexes on
  DNA
• Many sets of grps bound simultaneous to effect
  transcription

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How Genetic Switches Work

• Simple regulatory modules theme of complex gene
  regulatory control regions in mammals
• 5-10 coding capacity of mam genome GRPs
• Ea gene regulated by set of GRPs
• Ea protein is product of gene that is in turn
  regulated by set of other proteins
• Activity of GRPs regulated

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How Genetic Switches Work

• Regulation of Activity of GRPs

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How Genetic Switches Work

• Human ß-globin Gene
• Complex regulation- 2 step process
• Expressed only in RBC at specific time during
  development
• Possesses own set of GRPs but also under control
  of LCR
• Cells where no globin gene expressed gene cluster
  tightly pkged
• Higher order pkging decondensed in RBS

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How Genetic Switches Work

• LCR regulatory seq that govern accessibility and
  expression of distant genes or gene clusters
• ß-thalassemia deletion in ß-globin LCR causing
  gene to remain transcriptionally silent
• Many LCRs present in human genome

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How Genetic Switches Work

• Insulators or Boundary Sequences
• Bind Specialized Proteins
• Regulatory compartmentalization (Define domains
  of gene expression)
• Buffer genes from repressing effects of
  heterochromatin
• Block effect of enhancers (insulator must be btwn
  enhancer and promoter)
• Mechanism not understood

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How Genetic Switches Work

• Bacteria use interchangeable sigma subunits to
  help regulate transcription while eucaryotes use
  (3) diff RNA Pol

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How Genetic Switches Work

• Procaryotes vs Eucaryotes?

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Molecular Genetic Mechanisms of Specialized
Cell Types

• Cell Memory prerequisite for the creation of
  organized tissues and the maintenance of stably
  differentiated cell types

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Molecular Genetic Mechanisms of Specialized
Cell Types

• Gene Expression and Specialized Cell Types
• Environmental effects
• Cell memory
• Logic circuits
• differentiate
• keep time
• remember events of the past
• adjust gene expression over whole chromosome

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Molecular Genetic Mechanisms of Specialized
Cell Types

• DNA rearrangements mediate phase variation in
  bacteria
• Site Specific Recombination at promoter

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Molecular Genetic Mechanisms of Specialized
Cell Types

• Rearrangements at the Mat locus determines
• mating type in budding yeast

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Molecular Genetic Mechanisms of Specialized
Cell Types
Positive Feedback Loops Involving GRPs can Create
Cell Memory
Lambda Repressor and Cro GRPs Maintain Mode of
Growth of Lambda Phage
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Molecular Mechanisms of Specialized Cell Types

• Heritable State of Bacteriophage Lambda
• Switch controls flip-flop btwn lytic and
  lysogenic state
• Governed by two proteins that repress ea others
  synthesis
• Lambda repressor protein cI
• Cro
• 50 genes in genome

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Molecular Mechanisms of Specialized Cell Types

• Lysogenic- bacteriophage DNA integrated into host
  genome
• Lytic- virus multiplies, capsid protein
  translated and encapsulates virus which exits
  host cell and in so doing lysis cell

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Molecular Mechanisms of Specialized Cell Types
Prophage or lysogenic state lambda repressor
occupies operator synthesis of Cro and
its own synthesis Lytic State Cro occupies diff
site on operator synthesis of cI and
synthesis its own synthesis to multiply and exit
host
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Molecular Mechanisms of Specialized Cell Types

• Internal rhythms
• Governs behavior at diff times of day
• Established by day/night cycle
• Operates via transcriptional feedback loop
• Resetting clock destruction of a key GRP

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Molecular Mechanisms of Specialized Cell Types

• Combinatorial control
• Expression of set of genes can be coordinated by
  single protein
• Effect of single GRP can be decisive

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Molecular Mechanisms of Specialized Cell Types

• Expression of critical GRP can trigger
  expression of entire battery of downstream genes
• Ability to switch many genes on or off
  coordinately impt to cell differentiation
• Conversion of one cell type to another by single
  GRP emphasizes how dramatic differences in cell
  types in size, shape, chemistry and function can
  be produced by differences in gene expression

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Molecular Mechanisms of Specialized Cell Types
Combinatorial Gene Control Creates Many
Different Cell Types in Eucaryotes
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Molecular Mechanisms of Specialized Cell Types
Combinatorial Gene Control Creates Many
Different Cell Types in Eucaryotes
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Molecular Mechanisms of Specialized Cell Types

• Formation of Entire Organ Coordinated by Single
  GRP
• Ey coordinates development of Drosophilia eye

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Molecular Mechanism ofSpecialized Cell Types

• Transmitting Stable Patterns of Gene Expression
• Positive feedback loops GRP activates own
  expression
• Inhibiting expression an inhibitor to activate
  and maintain own expression
• Propagation of chromatin structure

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Molecular Mechanism ofSpecialized Cell Types

• Chromatin states
• heritable
• establish and preserve patterns of gene
  expression

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Molecular Mechanism ofSpecialized Cell Types

• Mechanisms of Dosage Compensation
• X-inactivation- humans
• Male specific up-regulation of transcription-
  Drosophilia
• Two-fold down regulation of X chromosome
  transcription- worm

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Molecular Mechanism ofSpecialized Cell Types
X-inactivation Center 106 nucleotide pairs Lg
regulatory center Seeds formation of
heterochromatin and facilitates its spread XIST
RNA coats inactive chromosome
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Molecular Mechanism ofSpecialized Cell Types

• Role of DNA Methylation in Gene Expression
• Patterns can be inherited
• Reinforces transcriptional repression established
  by other mechanisms
• Lock genes in silent state- preventing leaky
  transcription (106 )
• Maintains integrity of genome
• Genomic imprinting

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Molecular Mechanism ofSpecialized Cell Types
Genomic Imprinting When the expression of a gene
is dependent upon whether it is maternally or
paternally inherited
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Molecular Mechanism ofSpecialized Cell Types
Maternal CTCF binds to insulator preventing
enhancer from interacting w/ Igf2 gene no
expression Paternal methylation at insulator
site prevents CTCF binding allowing enhancer to
interact w/ Igf2 gene transcription
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Molecular Mechanism ofSpecialized Cell Types

• CG Islands
• Deamination of methylated Cs
  nonmutant T
• Deamination of methylated Cs U which
  is repaired
• Over evolutionary time 3 out of 4 CGs lost in
  this way
• Remaining CG unevenly distributed

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Posttranscriptional Regulation

• Posttranscriptional Controls
• Operate after RNA Pol initiated transcription
• Less common than transcriptional control but
• essential in many cases

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Posttranscriptional Regulation

• Transcriptional Attenuation
• Premature termination of transcription
• mRNA structure interacts w/ RNA Pol in manner
  that aborts transcription
• Premature termination can be prevented by
  proteins that bind to mRNA stem loop

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Posttranscriptional Regulation

• Alternative Splicing
• Different ways to splice primary transcript
  resulting in different polypeptides
• Protein complexity can exceed number of genes
• Regulation both positive and negative

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Posttranscriptional Regulation

• Regulation of RNA cleavage site and
  Poly-A-addition
• Changes COOH terminus
• Ex membrane bound or secreted antibody molecules
  by B lymphocytes

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Posttranscriptional Regulation

• RNA Editing
• Posttranscriptional alternation in mRNA sequence
• Tranpanosome mitochondrial sequences insertion
• of Us
• Plant mitochondrial genes Cs changed to Us
• Mediated by guide RNAs w/ 5 end comple-
• mentary to transcript
• Mammals deamination of adenine to inosine
• which pairs w/ C mediated by ADARs that
• recognize ds RNA structure

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Posttranscriptional Regulation

• Regulation of nuclear export