8' Control of Gene Expression 1

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8. Control of Gene Expression (1)
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Control of Gene Expression

• During differentiation cells begin to express a
  range of proteins
• Thus some genes are expressed while others are
  repressed
• Mammalian genome 30k protein coding genes
• But only 5k polypeptides manufactured at one time
• Regulation of gene expression is very complex
• Presently we have a superficial understanding

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Control of Gene Expression

• Synthesis of a protein involves discrete steps
• Several levels at which control mechanisms work
• Three main levels
• Transcription
• Processing of mRNA
• Translation

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Control of Gene Expression

• Transcriptional level control
• Differential gene transcription the major
  mechanism of selective protein synthesis
• Governed by a large number of proteins known as
  transcription factors
• Two functional classes of transcription factors
• General transcription factors
• Specific transcription factors
• Main topic of discussion

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Control of Gene Expression

• Specific transcription factors
• A single gene controlled by many regulatory sites
  bind different regulatory proteins
• A single regulatory protein may become attached
  to numerous sites on the genome
• Cells respond to environmental stimuli by
  synthesizing different transcription factors
• Bind to different sites on DNA
• Extent of transcription depends on the particular
  combination of transcription factors activated
  An example phosphoenolpyruvate carboxylase (PEPCK)

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Control of Gene Expression

• Specific transcription factors
• PEPCK
• A key enzyme of gluconeogenesis (conversion of
  pyruvate to glucose)
• Synthesized in liver in response to low glucose
• Synthesis drops sharply after a meal
• Level of synthesis of PEPKC controlled by
  different transcription factors
• eg receptors for hormones involved in regulating
  carbohydrate metabolism

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Control of Gene Expression

• Specific transcription factors
• PEPCK
• Closest upstream sequence
• TATA box major element of the genes promoter
• Region from TATA box to start of transcription
  site is the core promoter
• Site of assembly of preinitiation complex RNA
  polymerase II and general transcription factors
• Two other promoter sequences
• CAAT box
• GC box

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Control of Gene Expression

• Specific transcription factors
• PEPCK

Core promoter
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Control of Gene Expression

• Specific transcription factors
• PEPCK
• TATA box determines the site of initiation of
  transcription
• CAAT and GC boxes regulate the frequency of
  transcription
• All located within 100 to 150 base pairs upstream
  of the transcription start site proximal
  promoter elements

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Control of Gene Expression

• Activation of Transcription
• Hormones which affect transcription of PEPCK
  include insulin, thyroid hormone, glucagon and
  glucocorticoids
• All affect transcription factors which bind DNA
• DNA sites bound by transcription factors are
  termed response elements
• Glucocorticoids stimulate PEPCK expression by
  binding to a specific DNA sequence termed a
  glucocorticoid response element (GRE)

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Control of Gene Expression

• Activation of Transcription
• Same GRE is located upstream from different genes
  on different chromosomes
• Thus a single stimulus elevated
  glucocorticoid concentrations simultaneously
  activates a range of genes needed in a
  comprehensive response to stress

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Control of Gene Expression

• Activation of Transcription
• Expression of genes also regulated by more
  distant DNA elements termed enhancers
• Can be experimentally moved without affecting
  their ability to enhance gene expression
• May be 1000s or 10000s base pairs upstream or
  downstream from the gene
• How??
• Brought into close proximity to the gene as DNA
  can form loops
• Promoters and enhancers cordoned off from other
  genes by sequences called insulators

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Control of Gene Expression

• Activation of Transcription
• Enhancers

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Control of Gene Expression

• Activation of Transcription
• Enhancers

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Control of Gene Expression

• Activation of Transcription
• A transcription factor bound to an enhancer may
  act via the following mechanisms
• Recruit general transcription factors and DNA
  polymerase II to the core promoter
• Stabilize the transcription machinery located in
  the core promoter
• Via an intermediary termed a coactivator
• Coactivators are large complexes with 15 to 20
  subunits
• Do not directly bind DNA
• Interact with a range of transcription factors

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Control of Gene Expression

• Structure of Transcription Factors
• Contain different domains which mediate the
  different functions at least two domains
• DNA-binding domain
• Activation domain
• Commonly form dimers
• Example
• Glucocorticoid receptor
• Binds DNA at the glucocorticoid response element
  (GRE)
• Ligand-binding domain / DNA-binding domain /
  Activation domain

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Control of Gene Expression

• Structure of Transcription Factors
• GRE
• A palindrome
• Two-fold nature is important
• Pairs of GR polypeptides bind to DNA forming
  dimers

5-AGAACAnnnTGTTCT-3 3-TCTTGTnnnACAAGA-5
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Control of Gene Expression

• Transcription Factor Motifs
• Transcription factors belong to each of several
  classes based upon specific types of binding
  domains or motifs
• Many contain an a-helix which is inserted into
  the DNA major groove
• Recognizes the particular nucleotide sequence
  lining the groove
• Binding between aa and DNA (including DNA
  backbone) via
• Van der Waals (hydrophobic) forces
• Ionic bonds
• And hydrogen bonds

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Control of Gene Expression

• Transcription Factor Motifs
• Common transcription factor motifs
• Zinc finger
• Helix-loop-helix
• Leucine zipper
• HMG box
• Shared feature
• Structurally stable framework
• Specific DNA recognizing sequences are correctly
  positioned

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Control of Gene Expression

• Transcription Factor Motifs
• Zinc finger
• Zn ion coordinated to two cysteines and two
  histidines
• Each contains multiple zinc finger domains

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Control of Gene Expression

• Transcription Factor Motifs
• Helix-loop-helix (HLH)
• Two a helices separated by a loop
• Often preceded by a stretch of basic aa which
  interact with a specific nucleotide string
• Always occur as dimers
• heterodimers

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Control of Gene Expression

• Transcription Factor Motifs
• Leucine zipper motif
• Leucines every seven aa along an a-helix
• All leucines face the same direction
• Two a-helices can zip together forming a coiled
  coil
• Basic aa on opposite side of coils