Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics
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Ch.22 Activating Transcription
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Eukaryotic Transcription

• Response Elements
• Transcription Factor Structure/Function
• Regulation of TF activity

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Control of gene expression

• Activation of chromatin structure
• Initiation of txn
• Txn processing
• Transport to cytoplasm
• Translation

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Activating txn

• Basal factors
• Bind to the start point together with RNA pol.
• Activators
• Bind to upstream short sequences
• Ubiquotious
• Specifically expresses/activated
• Coactivators
• Establish contact between the TFs and the
  holocomplex
• Chromatin remodeling

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Response Elements

• Genes under common control
• Share common sequence elements recognized by a
  regulatory TF
• Termed response element
• Analogous to upstream elements
• Many different types

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Response Elements

• Many different types
• HSE - heat shock element
• MRE - metallothione response element
• GRE - glucocorticoid response element
• BLE - basal level element
• TRE - Phorbol ester response element

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Response Elements

• Specific regulated TFs interact with the upstream
  response elements
• HSE - Heat shock TF
• TRE - AP1 TF
• BLE - AP2 TF
• GRE - steroid receptor

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Fig 22.11 Regulatory region of a human
metallothionein gene
Also reinforces concept of modularity of
promoters
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Eukaryotic Transcription

• Response Elements
• Transcription Factor Structure/Function
• Regulation of TF activity

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Transcription Factor Structure/Function

• Multiple domains on TFs
• DNA binding domains
• Protein-Protein interacting domains
• Dimerization
• Activation

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Fig 22.3 Different domains for DNA binding and
activating
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TF Structure / Function

• Is DNA binding required for activation?
• Is only the activation domain sufficient?

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Gal 4 functional regions
Regulates genes encoding proteins involved in
the metabolism of galactose
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Fig 24.4 Txn activation by Gal 4
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Fig 22.5 Txn activation by tat
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TF Structure/Function

• DNA binding domains v. important
• Protein-Protein interactions also important
• May not need to be bound to DNA
• May bind to other TFs bound to DNA

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Two hybrid system
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The two- hybrid system

• Activation occurs only if the activation domain
  is brought to the promoter via protein
  interactions

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Fig 22.7 Role of co-activators
DNA - Protein Interactions Protein-Protein
Interactions
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Fig 22.8 Activators co-activators
May act at different stages of initiation
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Role of coactivators

• Increase binding of RNA Pol II to promoter
• Induces change in basal txn complex which
  increases its efficiency
• Induces changes in chromatin
• (chromatin remodeling)

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Activators and coactivators

• Txn activation domain makes contact with basal
  txn apparatus
• Influence the assembly of basal apparatus
• TFIID, TFIIB or TFIIA
• Most common interaction with TAFs (TFIID)
• Interaction with TFIIB
• Enhances binding of TFIIB to the promoter

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How does the activator stimulate txn?

• 1-Recruites RNA pol complex, to increase its
  binding to the promoter
• 2-Induces changes in the txn complex, that would
  increase its efficiency
• Changes in enzyme conformation
• Alter chromatin structure

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RNA Pol binds as an holoenzyme

• Fig 22.9

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Transcription factors regulate gene expresssion

• Fig. 22.10

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Eukaryotic Transcription

• Response Elements
• Transcription Factor Structure/Function
• Regulation of TF activity

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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Zinc finger TFs

• Several different types of Zn fingers
• Cys2-His2
• Cys2- Cys2 (steroid receptor)
• Cys6
• Cys2- Cys2 (GATA-1 like)

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Cys2-His2 Zn finger

• Very common DNA binding domain
• Consensus sequence
• Cys - Xaa2-Cys-Xaa13-Cys-Xaa2-Cys
• Originally found in TFIIIA, now recognized in
  many TFs

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Fig 22.13 Three Zn fingers of TF SP1
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Fig 22.14 DNA binding by Zn fingers
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Steroid receptor Zn fingers

• Cys2-Cys2
• Distinct from Cys2-His2
• Consensus sequence
• Cys - Xaa2-Cys-Xaa13-Cys-Xaa2-Cys
• Zn atom bound by 4 Cys

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Fig 22.17-18 Steroid receptor Zn fingers
First Finger
ERE
GRE
Fingers are not equivalent!
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Cys6 Zn Finger

• Gal 4 and many other yeast TFs
• Consensus binding site
• Gal4 CGG N11 CCG
• PPR1 CGG N6 CGG

Adjusting linker length alters binding site
Cys
Cys
Zn
Cys
Cys
Zn
Zn
Zn
CGG N6-11 CCG
Cys
Cys
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GATA-1 Like Zn Finger

• GATA 1,2,3,4
• erythrocyte gene expression and maturation
• globin genes
• Consensus sequence
• Cys-Xaa2-Cys-Xaa17-Cys-Xaa2-Cys (29aa)
• Two Zn fingers

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GATA-1 Like Zn Finger

• 8bp DNA binding site
• binds as a monomer
• Zn binding domain not required for specific DNA
  binding
• Only 2nd finger and next 28 residues

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Steroid Hormones

• Synthesized in response to various neuroendocrine
  activities
• gt30 types secreted by adrenal gland
• Regulate body development and function by
  regulating gene expression

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Steroid Hormones

• 2 major groups of corticoids
• Glucocorticoids cortisol
• Mineralcorticoids aldosterone
• Sex hormones
• Estrogens b-estradiol
• Androgens testosterone

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Steroid Hormones

• Others
• Morphogenesis (trans) retinoic acid
• Development vitamin D3
• Thyroid hormones triiodothyronine (T3)

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Fig 21.6 Steroid Hormone Structures
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Steroid hormones

• Specific
• Each compound affects specific pathways of gene
  expression
• Common
• Each compound is a small molecule, binds to a
  specific receptor, activates specific
  genes/pathways
• Steroid receptor -gt Transcription factor

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Fig 22.16 Organization of steroid receptors
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Fig 22.19 Function of glucocorticoid receptors

• At least two major domains
• DNA binding txn activation
• Steroid binding

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Steroid Hormone Receptor TFs

• 2 Groups based on dimerization
• Homodimer forming
• Heterodimer forming

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Homodimer forming steroid receptor TFs

• Palindromic response element
• Half site is TGTTCT
• Receptors for
• Glucocorticoid
• Mineralocorticoid
• Androgen
• Progesterone
• Estrogen (Half site is TGACCT)

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Heterodimer forming steroid receptor TFs

• Response element
• Direct repeat of two half-sites
• Half site is TGACCT
• Recognition is based on separation of repeats
• TGACCT N1-5 TGACCT

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Heterodimer forming steroid receptor TFs

• Receptors for Separation
• Thyroid (T3R) 4bp
• Vitamin D (VDR) 3bp
• Retinoic Acid (RAR) 5bp
• 9-cis retinoic Acid (RXR) 1bp
• (where RXR is one of the subunits)

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Steroid receptors act as dimers
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Fig 22.22 Txn activation by Steroid Receptor TFs
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Fig 22.22 Txn activation by Steroid Receptor TFs
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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Helix-Turn-Helix (Homeodomain) TFs

• DNA binding domain
• two short a-helices separated by short stretch of
  amino acids (turn)
• one helix lies in major groove, other lies across
  the helix at an angle

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Helix-Turn-Helix (Homeodomain) TFs

• First identified in phage repressors
• cI, cro proteins
• Homeodomain containing Euk. TFs have a similar
  structure
• 60 aa segment
• found in proteins regulating early development

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Fig 22.23 Homeodomain TFs
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Fig 22.24 Conserved nature of homeodomain region
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Fig 22.25 DNA binding by Homeodomain
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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Helix-Loop-Helix TFs

• Two a-helices joined by a loop
• 40-50 aa in size
• amphipathic a-helices
• loop is of variable size

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Helix-Loop-Helix TFs

• Form dimers (hetero- and homo-)
• basic region near dimerization domain
• bHLH

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Fig. 22.26 Helix-Loop-Helix TFs
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Helix-Loop-Helix TFs

• Two groups (mammalian TFs)
• Class A
• ubiquitously expressed
• e.g., mammalian E12/E47
• Class B
• tissue-specific expression
• e.g., MyoD, myogenin, Myf-5

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Helix-Loop-Helix TFs

• Others
• Drosophila
• Ac-S (tissue-specific) / da (ubiquitous)
• Mammaliam
• Myc (proto-oncogenes)
• different partners and targets

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Helix-Loop-Helix TFs

• Heterodimers
• often form between Class A and Class B
• effects DNA binding ability

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Helix-Loop-Helix TFs

• Dimer partner effects DNA binding
• Dimer dimerization DNA binding
• E47/E47 good high affin.
• E12/E12 poorly low affin.
• MyoD/MyoD poorly low affin.
• E12/E47 good high affin.
• MyoD/E47 good high affin.

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Helix-Loop-Helix TFs

• Cant predict ability to dimerize or DNA binding
  ability
• all that bind recognize the same DNA seq.

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Helix-Loop-Helix TFs

• Some HLH proteins lack the basic region
• e.g., Id (mammalian), emc (Drosophila)

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Fig 22.27
Id regulates myogenesis by preventing formation
of functional MyoD/E12(E47) dimer
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Helix-Loop-Helix TFs

• Two general principles of Txn regulation
  illustrated by HLH TFs
• Combinatorial associations of small numbers of
  proteins. Different combinations may have
  different functions
• Some HLH TFs lack basic region for DNA binding -
  function as suppressors

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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Leucine Zipper

• amphipathic a-helix like HLH
• every 7th aa in helix is Leucine
• L X6 L X6 L X6 L X6 L X6 L
• generally at least 4 Leu in zipper

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Leucine Zipper

• Helices are dimerization site
• forms a coiled - coil
• basic region at end of helix
• bZip TFs

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Fig 22.28 Leucine Zipper
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Leucine Zipper

• Can form hetero- or homodimers
• C/EBP homodimers CCAAT box
• jun-fos heterodimers AP1 TF
• jun homodimers 10-fold lower
• fos no homodimers

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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Activation Domains

• Several different domains can function as
  transcriptional activators
• Acidic
• Glutamine rich
• Proline rich

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Activation Domains

• Acidic
• high content of Asp (D) and Glu (E)
• no defined structure (possibly b-sheet)
• Gal4, GCN4, VP16
• in GCN4 - 18/88 aa are acidic - 2 basic

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Activation Domains

• Glutamine-rich
• high content of Glutamine (Gln, Q)
• in SP1, 2 regions contain about 25 Gln
• No sequence homology
• overall character of domain important
• found in Sp1, Oct12, Drosophila homeobox TFs,
  some yeast TFs

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Activation Domains

• Proline rich
• high content of Proline (Pro, P)
• 19/84 aa 23
• no sequence homology - overall character
• foun in jun, AP2, Oct2, CTF/NF1

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TF Structure / Function

• Many structural domains in TFs required for
  activity
• Zn finger
• Steroid receptor
• helix-turn-helix (homeodomain)
• helix-loop-helix
• leucine zipper (b-zip)
• activation domains

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Eukaryotic Transcription

• Response Elements
• Transcription Factor Structure/Function
• Regulation of TF activity

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How can activity of TFs be regulated?

• Obvious mechanism is at level of transcription
  and translation
• Other mechanisms?

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Fig 22.12 Control of activity of TFs
AP1
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Fig 22.12 Control of activity of TFs
I-kB
Id
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Eukaryotic Transcription

• Response Elements
• Transcription Factor Structure/Function
• Regulation of TF activity