Section N Regulation of transcription in eukaryotes

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Section N Regulation of transcription in
eukaryotes

• Molecular Biology Course

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N1 Eukaryotic Transcription Factors

• 1. Transcription factor domain structure
• (DNA-binding, dimerization, transcription
  activation, repressor)
• 2. Targets for transcriptional regulation
  

N2 Examples of transcriptional regulation
SP1, hormonal regulation, phosphorylation of STAT
proteins, HIV Tat, myoD homeodomain proteins
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N1 Eukaryotic Transcription Factors
Section N Regulation of transcription in
eukaryotes

• Transcription factor domain structure (link)
• DNA-binding domains
• Dimerization domains
• Transcription activation domains
• Repressor domains
• Targets for transcriptional regulation (link)

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Transcription of a single gene may be regulated
by many different factors interacting with
regulatory elements upstream or downstream of the
transcribed sequence.
Start site
Gene X
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Regulatory elements to bind transcription factors
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Example The metallothionein (MT, ?????) gene
The metallothionein protein protects the cell
against excess concentrations of heavy metals, by
binding the metal and removing it from the cell.
The gene is expressed at a basal level, but is
induced to greater levels of expression by heavy
metal ions (such as cadmium) or by
glucocorticoids (????).
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• Common features of transcription factors
• bind specifically to some DNA sites specific
  motifs in promoters, upstream regulatory elements
  (UREs) or enhancer regions. Some factors modulate
  transcription by protein-protein intracation
• Activate/repress transcription.

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Transcription factors domain structure
Transcription factor Pdr1
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- The activity of a transcription factor can be
assigned to separate protein domains

• activation domains. (activity)
• DNA-binding domains. (activity)

• dimerization domains. Many transcription factors
  occur as homo- or heterodimers, held together by
  dimerization domains. (regulation)
• ligand-binding domains. Allowing regulation of
  transcription factor activity by binding of an
  accessory small molecule.The steroid hormone
  receptors are an example containing all for of
  these types of domain. (regulation)

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Domain swap experiments moving domains among
proteins, proving that domains can be dissected
into separate parts of the proteins.
The experiment of fusing activation domains of
yeast transcription factors Gal4 and Gcn4 into
the bacterial LexA repressor is described in your
text book. Transcription activation domains are
separable from their DNA binding activity.
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Another example construction of new proteins
capable of binding to DNA
NLS nucleus localization signal
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N1-2 DNA-binding domains

• The helix-turn-helix domain
• The zinc finger domain
• The basic domain
• Return to menu

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The helix-turn-helix domain
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Examples of Helix-turn-helix domains

• 1. Homeodomain encoded by a sequence called the
  homeobox, containing a 60-amino-acid. In the
  Antennapedia transcription factor of Drosophila,
  this domain consists of four a-helices in which
  helices ?and ? are at right angles to each other
  and are separated by a characteristic ß-turn.

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• 2. Bacteriophage DNA-binding proteins such as the
  phage ? cro repressor, lac and trp repressors,
  and cAMP receptor protein, CRP.

• The recognition helix of the domain structure
  lies partly in the major groove and interacts
  with the DNA.
• The recognition helices of two homeodomain
  factors Bicoid and Antennapedia can be exchanged,
  and this swaps their DNA-binding specificities.

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The zinc finger domain
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• Zinc finger domain exists in two forms.
• C2H2 zinc finger a loop of 12 amino acids
  anchored by two cysteine and two histidine
  residues that tetrahedrally co-ordinate a zinc
  ion. This motif folds into a compact structure
  comprising two ß-strands and one a-helix. The
  a-helix containing conserved basic amino acids
  binds in the major groove of DNA
  (picturepicture2)

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• Examples
• (1) TFIIIA, the RNA Pol III transcription
  factor, with C2H2 zinc finger repeated 9 times.
• (2) SP1, with 3 copies of C2H2 zinc finger.
• Usually, three or more C2H2 zinc fingers are
  required for DNA binding.

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2. C4 zinc finger zinc ion is coordinated by 4
cysteine residues. Example steriod hormone
receptor transcription factors (N2) consisting of
homo- or hetero-dimers, in which each monomer
contains two C4 zinc finger. (picture)
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The basic domain

• Rich in basic amino acid residues
• found in a number of DNA-binding proteins
• generally associated with one or other of two
  dimerization domains, the leucine zipper or the
  helix-loop-helix(HLH) motif, resulting in basic
  leucine zipper (bZIP) or basic HLH proteins.
  Dimerization of the proteins brings together two
  basic domains which can then interact with DNA.

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N1-3 Dimerization domains

• Leucine zippers
• The helix-loop-helix domain (HLH)

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Leucine zippers

• Leucine zipper proteins contain a hydrophobic
  leucine residue at every seventh position in a
  region that is often at the C-terminal part of
  the DNA-binding domain (picture.).
• These leucines are responsible for dimerization
  through interaction between the hydrophobic faces
  of the a-helices. This interaction forms a
  coiled-coil structure

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• bZIP (basic leucine zipper) transcription
  factors contain a basic DNA-binding domain
  N-terminal to the leucine zipper. The N-terminal
  basic domains of each helix form a symmetrical
  structure in which each basic domains lies along
  the DNA in opposite direction, interacting with a
  symmetrical DNA recognition site with the
  zippered protein clamp (pic1..)
• The leucine zipper is also used as a dimerization
  domain in proteins containing DNA-binding domains
  other than the basic domain, including some
  homeodomain proteins.

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The helix-loop-helix domain (HLH)

• The overall structure is similar to the leucine
  zipper, except that a nonhelical loop of
  polypeptide chain separates two a-helices in each
  monomeric protein.
• Hydrophobic residues on one side of the
  C-terminal a-helix allow dimerization.
• Example MyoD (pic..) family of proteins.

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• Similar to leucine zipper, the HLH motif is often
  found adjacent to a basic domain that requires
  dimerization for DNA binding.
• Basic HLH proteins and bZIP proteins can form
  heterodimers allowing much greater diversity and
  complexity in the transcription factor repertoire.

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N1-4 Transcription activation domains

• Acidic activation domains
• Glutamine-rich domains
• Proline-rich domains

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Acidic activation domains

• Also called acid blobs or negative noodles
• Rich in acidic amino acids
• Exists in many transciption activation domains
• yeast Gcn4 and Gal4,
• mammalian glucocorticoid receptor
• herpes virus activator VP16 domains.

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Glutamine-rich domains

• Rich in glutamine
• the proportion of glutamine residued seems to be
  more important than overall structure.
• Exists in the general transcription factor SP1.

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Proline-rich domains

• Proline-rich
• continuous run of proline residues can activate
  transcription
• Exists in transcription factors c-jun, AP2 and
  Oct-2.

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N1-5 Repressor domains

• Repression of transcription may occur by indirect
  interference with the function of an activator.
  This may occur by
• Blocking the activator DNA-binding site (as with
  prokaryotic repressors, wrong)
• Formation of a non-DNA-binding complex (e.g. the
  Id protein which blocks HLH protein-DNA
  interactions, since it lacks a DNA-binding
  domain, N2).

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• 3. Masking of the activation domain without
  preventing DNA binding (e.g. Gal80 masks the
  activation domain of the yeast transcription
  factor Gal4).
• A specific domain of the repressor is directly
  responsible for inhibition of transcription.
  (e.g. prokaryotic repressors)
• e.g. A domain of the mammalian thyroid hormone
  receptor can repress transcription (page 212
  214).
• Return to menu

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N1-6 Targets for transcriptional regulation
(pic)

• chromatin structure
• interaction with TFIID through specific TAFIIS
• interaction with TFIIB
• interaction or modulation of the TFIIH complex
  activity leading to differential posphorylation
  of the CTD of RNA Pol II.

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• It seems likely that different activation domains
  may have different targets, and almost any
  component or stage in initiation and
  transcription elongation could be a target for
  regulation resulting in multistage regulation of
  transcription.Return to menu

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Section N Regulation of transcription in
eukaryotes
N2 Examples of transcriptional regulation
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• Constitutive transcription factorsSP1
• Hormonal regulationsteroid hormone receptors
• Regulation by phosphorylationSTAT proteins
• Transcription elongationHIV Tat
• Cell determinationmyoD
• Embryonic developmenthomeodomain proteins

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N2-1 Constitutive transcription factorsSP1

• binds to a GC-rich sequence with the consensus
  sequence GGGCGG.
• binding site is in the promoter of many
  housekeeping genes
• It is a constitutive transcription factor present
  in all cell types.
• contains three zinc finger motifs and two
  glutamine-rich activation domains interacting
  with TAFII110, thus regulating the basal
  transcription complex.

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N2-2 Hormonal regulationsteroid hormone
receptors

• Many transcription factors are activated by
  hormones which are secreted by one cell type and
  transmit a signal to a different cell type.
• steroid hormones lipid soluble and can diffuse
  through cell membranes to interact with
  transcription factors called steroid hormone
  receptors.

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• In the absence of steroid hormone, the receptor
  is bound to an inhibitor, and located in the
  cytoplasm (picture).
• In the presence of steroid hormone,
• the hormone binds to the receptor and releases
  the receptor from the inhibitor,
• receptor dimerization and translocation to the
  nucleus.
• receptor interaction its specific DNA-binding
  sequence (response element) via its DNA-binding
  domain, activating the target gene.

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• Steroid hormones involving important hormone
  receptors glucocorticoid (?????), estrogen
  (???), retinoic acid (???)and thyroid hormone
  (?????)receptors.

Please noted that the above model is not true for
all these hormone receptors

• Thyroid hormone receptor is a DNA-bound repressor
  in the absence of hormone, which converted to a
  transcriptional activator.

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N2-3 Regulation by phosphorylation STAT proteins

• For hormones that do not diffuse into the cell.
• The hormones binds to cell-surface receptors and
  pass a signal to proteins within the cell through
  signal transduction.
• Signal transduction often involves protein
  phosphorylation.
• Example Interferon-? induces phosphorylation of
  a transcription factor called STAT1a through
  activation of the intracellular kinase called
  Janus activated kinase(JAK). go on...

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• Unphosphorylated STAT1a protein exists as a
  monomer in the cell cytoplasm and has no
  transcriptional activity.
• Phosphorylated STAT1a at a specific tyrosine
  residue forms a homodimer which moves into the
  nucleus to activate the expression of target
  genes whose promoter regions contain a consensus
  DNA-binding motif (picturepic3..)

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N2-4 Transcription elongationHIV Tat

• Human immunodeficiency virus (HIV)(pic) encodes
  an activator protein called Tat, which is
  required for productive HIV gene
  expression(pic..).
• Tat binds to an RNA stem-loop structure called
  TAR, which is present in the 5-UTR of all HIV
  RNAs just after the HIV transcription start site,
  to regulate the level of transcription
  elongation.

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• In the absence of Tat, the HIV transcripts
  terminate prematurely due to poor processivity of
  the RNA Pol ? transcription complex.
• Tat binds to TAR on one transcript in a complex
  together with cellular RNA-binding factors. This
  protein-RNA complex may loop backwards and
  interact with the new transcription initiation
  complex which is assembled at the promoter. go
  on...

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• This interaction may result in the activation of
  the kinase activity of TFIIH, leading to
  phosphorylation of the carboxyl-terminal domain
  (CTD) of RNA Pol?, making the polymerase a
  processive enzyme to read through the HIV
  transcription unit, leading to the productive
  synthesis of HIV proteins (picture..)

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N2-5 Cell determinationmyoD (pic1..pic2..)

• myoD was identified as a gene to regulate gene
  expression in cell determination, commanding
  cells to form muscle.
• MyoD protein has been shown to activate
  muscle-specific gene expression directly.
  Overexpression of myoD can turn fibroblasts into
  muscle-like cells which express muscle-specific
  genes and resemble myotomes.
• myoD also activates expression of p21waf1/cip1
  expression, a small molecule inhibitor of CDKs,
  causing cells arrested at the G1-phase of the
  cell cycle which is characteristic of
  differentiated cells. .

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• Four genes,myoD,myogenin, myf5 and mrf4 have been
  shown to have the ability to convert fibroblasts
  into muscle. The encoded proteins are all members
  of the helix-loop-helix (HLH for dimerization)
  transcription factor family.
• These proteins are regulated by an inhibitor
  called Id that lacks a DNA-binding domain, but
  contains the HLH dimerization domain. Id protein
  can bind to MyoD and related proteins, but the
  resulting heterodimers cannot bind DNA, and hence
  cannot regulate transcription

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N2-6 Embryonic development homeodomain proteins

• The homeobox is a conserved DNA sequence which
  encodes the helix-turn-helix DNA binding protein
  structure called the homeodomain.
• Homeotic genes of Drosophila are responsible for
  the correct specification of body parts. For
  example, mutation of one of these genes,
  Antennapedia, causes the fly to form a leg where
  the antenna should be.
• conserved between a wide range of eukaryotes.
• important in mammalian development.

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• 1. TFIID
• multiprotein complex
• including TBP, other proteins are known as
  TAFIIs
• TBP is the only protein binds to TATA box

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• 3. TFIIB RNA Pol binding
• binds to TFIID
• Binds to RNA Pol with TFIIF

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5. phosphorylation of the polymerase CTD by
TFIIH Formation of a processive RNA polymerase
complex and allows the RNA Pol to leave the
promoter region.
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HIV genome

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