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Section N Regulation of transcription in eukaryotes

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Title: Section N Regulation of transcription in eukaryotes


1
Section N Regulation of transcription in
eukaryotes
  • Molecular Biology Course

2
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
3
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)

4
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
1
Regulatory elements to bind transcription factors
5
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 (????).
6
  • 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.

7
Transcription factors domain structure
Transcription factor Pdr1
8
- 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)

9
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.
10
Another example construction of new proteins
capable of binding to DNA
NLS nucleus localization signal
11
N1-2 DNA-binding domains
  • The helix-turn-helix domain
  • The zinc finger domain
  • The basic domain
  • Return to menu

12
The helix-turn-helix domain
13
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.

14
  • 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.

15
The zinc finger domain
16
  • 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)

17
  • 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.

18
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)
19
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.

20
N1-3 Dimerization domains
  • Leucine zippers
  • The helix-loop-helix domain (HLH)

21
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

22
  • 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.

23
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.

24
  • 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.

25
N1-4 Transcription activation domains
  • Acidic activation domains
  • Glutamine-rich domains
  • Proline-rich domains

26
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.

27
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.

28
Proline-rich domains
  • Proline-rich
  • continuous run of proline residues can activate
    transcription
  • Exists in transcription factors c-jun, AP2 and
    Oct-2.

29
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).

30
  • 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

31
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.

32
  • 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

33
Section N Regulation of transcription in
eukaryotes
N2 Examples of transcriptional regulation
34
  • Constitutive transcription factorsSP1
  • Hormonal regulationsteroid hormone receptors
  • Regulation by phosphorylationSTAT proteins
  • Transcription elongationHIV Tat
  • Cell determinationmyoD
  • Embryonic developmenthomeodomain proteins

35
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.

36
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.

37
  • 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.

38
  • 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.

39
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...

40
  • 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..)

41
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.

42
  • 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...

43
  • 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..)

44
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. .

45
  • 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

46
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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