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Section Prokaryotic and Eukaryotic Chromosome Structure

VIEWS: 66 PAGES: 46

									 Section N – Regulation of
transcription in eukaryotes
              Contents

N1 Eukaryotic transcription factors
 Transcription factor domain structure, DNA-
 binding domains, Dimerization domains,
 Transcription activation domains, Repressor
 domains, Targets for transcriptional regulation
N2 Eukaryotic of transcriptional
 regulation
 Constitutive transcription factors:SP1, Hormonal
 regulation: steroid hormone receptors, Regulation
 by phosphorylation: STAR proteins, Transcription
 elongation: HIV Tat, Cell determination: myoD,
 Embrynic development: homeodomain proteins
N1 Eukaryotic transcription factors —
Transcription factor domain structure
  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
N1 Eukaryotic transcription factors —
                   DNA- binding domains



   1. The helix-turn-helix domain
   2. The zinc finger domain
   3. The basic domain
1. The helix-turn-helix domain
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 α-helices in
  which helices Ⅱand Ⅲ are at right
  angles to each other and are separated
  by a characteristic β-turn.
2). Bacteriophage DNA-binding proteins
  such as the phage λ 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.
2. The zinc finger domain
   Zinc finger domain exists in
    two forms.
①   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 β-strands
    and one α-helix. The α-helix
    containing conserved basic amino
    acids binds in the major groove of
    DNA
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.
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.
N1 Eukaryotic transcription factors —
                     Dimerization domains


      • Leucine zippers
      • The helix-loop-helix domain
  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 .
• These leucines are responsible for
  dimerization through interaction
  between the hydrophobic faces of the
  α-helices. This interaction forms a
  coiled-coil structure
• 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

• 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.
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 α-helices in each
    monomeric protein.
  • Hydrophobic residues on one side of
    the C-terminal α-helix allow
    dimerization.
  • Example: MyoD family of proteins.
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.
N1 Eukaryotic transcription factors —
     Transcription activation domains


1. Acidic activation domains
2. Glutamine-rich domains
3. Proline-rich domains
Acidic activation domains
• Also called “acid blobs” or
   “negative noodles”
• Rich in acidic amino acids
• Exists in many transciption
   activation domains
1. yeast Gcn4 and Gal4,
2. mammalian glucocorticoid
   receptor
3. herpes virus activator VP16
   domains.
 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.
 Proline-rich domains
• Proline-rich
• continuous run of proline
  residues can activate
  transcription
• Exists in transcription factors c-
  jun, AP2 and Oct-2.
N1 Eukaryotic transcription factors —
                       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).
• 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
N1 Eukaryotic transcription factors —
Targets for transcriptional regulation
• 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.
• 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.
N2 Eukaryotic of transcriptional regulation —
Constitutive transcription factors:SP1
• 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.
N2 Eukaryotic of transcriptional regulation —
           Hormonal regulation: steroid
                   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.
    In the absence of steroid hormone,
     the receptor is bound to an inhibitor,
     and located in the cytoplasm.
    In the presence of steroid hormone,
1.   the hormone binds to the receptor
     and releases the receptor from the
     inhibitor,
2.   receptor dimerization and
     translocation to the nucleus.
3.   receptor interaction its specific DNA-
     binding sequence (response element)
     via its DNA-binding domain,
     activating the target gene.
Steroidhormones involving
important hormone receptors:
glucocorticoid (糖皮质激素),
estrogen (雌激素), retinoic acid (视
黄酸)and thyroid hormone (甲状
腺激素)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.
N2 Eukaryotic of transcriptional regulation —
        Regulation by phosphorylation:
                        STAR 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-γ induces phosphorylation
  of a transcription factor called STAT1α
  through activation of the intracellular kinase
  called Janus activated kinase(JAK).
1. Unphosphorylated STAT1α protein:
   exists as a monomer in the cell
   cytoplasm and has no
   transcriptional activity.
2. Phosphorylated STAT1α 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
N2 Eukaryotic of transcriptional regulation —
     Transcription elongation: HIV 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.
• In the absence of Tat, the HIV
  transcripts terminate prematurely due to
  poor processivity of the RNA Pol Ⅱ
  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.
• This interaction may result in the
  activation of the kinase activity of TFIIH,
  leading to phosphorylation of the
  carboxyl-terminal domain (CTD) of RNA
  PolⅡ, making the polymerase a
  processive enzyme to read through the
  HIV transcription unit, leading to the
  productive synthesis of HIV proteins
N2 Eukaryotic of transcriptional regulation —
                 Cell determination: myoD
• 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. .
• 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
N2 Eukaryotic of transcriptional regulation —
                  Embrynic 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.
       Multiple choice questions
1. Which two of the following statements about transcription factors are true?
A  the helix-turn-helix domain is a transcriptional activation domain.
B  dimerization of transcription factors occurs through the basic domain.
C  leucine zippers bind to DNA.
D  it is often possible to get functional transcription factors when DNA binding domains
    and acti-vation domains from separate transcription factors are fused together.
E the same domain of a transcription factor can act both as a repressor and as an
    activation domain.
2. Which two of the following statements about transcriptional regulation are
    false?
A SP1 contains two adivation domains.
B steroid hormones regulate transcription through binding to cell surface receptors.
C phosphorylation of Stat1α leads to its migration from the cytoplasm to the nucleus.
D HIV Tat regulates RNA Pol II phosphorylation and processivity.
E the MyoD protein can form heterodimers with a set of other HLH transcription factors.
F the homeobox is a conserved DNA binding domain.
THANK YOU !

								
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