4' Eukaryotic Transcriptional Regulation

1
4. Eukaryotic Transcriptional Regulation
a). Characteristics of eukaryotic RNA and their
polymerases i). Classes of cellular RNA ii).
RNA polymerases I, II, and III b). Transcription
of messenger RNA in eukaryotes i). Structure of
eukaryotic messenger RNA ii). Complexity of
mRNA populations in the cell iii). Promoters
and transcription elements iv). Transcription
factors General transcription
factors Basic region-leucine zipper
proteins Zinc finger transcription
factors v). Mutations affecting promoters
2

• Classes of eukaryotic cellular RNAs
• ribosomal RNA (rRNA)
• 18S (small subunit)
• 28S (large subunit)
• 5.8S (large subunit)
• 5S (large subunit)
• transfer RNA (tRNA)
• messenger RNA (mRNA)
• heterogeneous nuclear RNA (hnRNA) (precursors of
  mRNA)
• small nuclear RNA (snRNA)
• U1, U2, U3, U4, U5, U6, U7, U8, U9, U10...
• small cytoplasmic RNA (scRNA)
• 7SL RNA
• What are the enzymes responsible for the
  synthesis of these RNAs?

3
The human RNA polymerases
Polymerase Location Product RNA polymerase I
nucleolus 18S, 28S, 5.8S rRNA RNA
polymerase II nucleoplasm
hnRNA/mRNA, U1, U2, U4, U5 snRNA
RNA polymerase III nucleoplasm tRNA,
5S RNA, U6 snRNA, 7SL RNA
mitochondrial RNA polymerase
mitochondrion all mitochondrial RNA
__________________________________________________
___________________________________________
Sensitivity of the nuclear RNA polymerases to
a-amanitin1 RNA pol I resistant RNA pol
II high sensitivity (binds with K 10-8 M)
RNA pol III low sensitivity (binds with K 10-6
M) 1 cyclic octapeptide from the poisonous
mushroom Amanita phalloides
4
Structure of eukaryotic mRNA
Cap
initiation
5 untranslated region
5
AUG
7mGpppN
translated region
UGA
termination
3 untranslated region
polyadenylation signal
3
(A)200
AAUAAA
poly(A) tail

• all mRNAs have a 5 cap and all mRNAs (with the
  exception
• of the histone mRNAs) contain a poly(A) tail
• the 5 cap and 3 poly(A) tail prevent mRNA
  degradation
• loss of the cap and poly(A) tail results in mRNA
  degradation

5

• Complexity1 of mRNA classes in the mammalian
  cell2
• Number of
• different
• Abundance Abundance mRNA
• class (copies/cell) species
  Total
• high 12,000 9
  108,000
• intermediate 300 700
  210,000
• low (rare) 15 11,500
  172,500
• 12,209 490,500
• Based on these measurements, this cell type
  contains
• three abundance classes of mRNA
• 12,209 different mRNA species
• 490,500 total mRNA molecules
• 1determined in RNA-DNA hybridization experiments
  analogous to Cot curves
• 2mouse liver cytoplasmic poly(A) RNA

6
Transcription and promoter elements for RNA
polymerase II
1
transcription element
exon
exon
P
TE
promoter

• Promoter (DNA sequence upstream of a gene)
• determines start site (1) for transcription
  initiation
• located immediately upstream of the start site
• allows basal (low level) transcription
• Transcription element (DNA sequence that
  regulates the gene)
• determines frequency or efficiency of
  transcription
• located upstream, downstream, or within genes
• can be very close to or thousands of base pairs
  from a gene
• includes
• enhancers (increase transcription rate)
• silencers (decrease transcription rate)
• response elements (target sequences for
  signaling molecules)
• genes can have numerous transcription elements

7
Transcription and promoter elements for RNA
polymerase II
transcription element
exon
exon
P
TE
promoter
transcription element
exon
exon
P
TE
promoter complex
exon
exon
P
TE
TE
exon
exon
P
TE
8
The locus control region is a specialized
transcription element
locus control region
LCR
TE
gene A
P
gene B
P
TE

• a single locus control region (LCR) may control
  two or more
• transcription units in a
  cell-specific fashion

9
Sequence elements within a typical eukaryotic
gene1
1 based on the thymidine kinase gene
octamer transcription element
1
promoter
GC
TATA
CAAT
GC
ATTTGCAT
-25
-50
-80
-95
-130

• TATA box (TATAAAA)
• located approximately 25-30 bp upstream of the
  1 start site
• determines the exact start site (not in all
  promoters)
• binds the TATA binding protein (TBP) which is a
  subunit of TFIID
• GC box (CCGCCC)
• binds Sp1 (Specificity factor 1)
• CAAT box (GGCCAATCT)
• binds CTF (CAAT box transcription factor)
• Octamer (ATTTGCAT)
• binds OTF (Octamer transcription factor)

10

• Proteins regulating eukaryotic mRNA
  synthesis
• General transcription factors
• TFIID (a multisubunit protein) binds to the TATA
  box
• to begin the assembly of the transcription
  apparatus
• the TATA binding protein (TBP) directly binds
  the TATA box
• TBP associated factors (TAFs) bind to TBP
• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH1, TFIIJ
  assemble with TFIID
• RNA polymerase II binds the promoter region via
  the TFIIs
• Transcription factors binding to other promoter
  elements and
• transcription elements interact with
  proteins at the promoter
• and further stabilize (or inhibit)
  formation of a functional
• preinitiation complex
• 1TFIIH is also involved in phosphorylation of RNA
  polymerase II, DNA repair
• (Cockayne syndrome mutations), and cell cycle
  regulation

11
Binding of the general transcription factors
F
TAFs
E
TFIID
B
H
TBP
A
J
1
-25

• TFIID (a multisubunit protein) binds to the TATA
  box
• to begin the assembly of the transcription
  apparatus
• the TATA binding protein (TBP) directly binds
  the TATA box
• TBP associated factors (TAFs) bind to TBP
• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ
  assemble with TFIID

12
Binding of RNA polymerase II
F
E
TFIID
B
H
TBP
A
J
RNA pol II

• RNA polymerase II (a multisubunit protein) binds
  to
• the promoter region by interacting with the
  TFIIs
• TFs recruit histone acetylase to the promoter

13
Binding of specialized TFs
F
E
TFIID
B
H
TBP
A
J
1
RNA pol II

• transcription factors binding to
• other promoter elements and transcription
  elements interact
• with proteins at the promoter and further
  stabilize (or inhibit)
• formation of a functional preinitiation
  complex
• this process is called transactivation

14
Formation of a stable preinitiation complex
F
E
B
TFIID
H
TBP
J
1
RNA pol II

• the stability and frequency with which complexes
  are formed
• determines the rate of initation of
  transcription
• the rate of initiation of transcription is of
  major importance in
• determining the abundance of an mRNA
  species

15
Initiation of transcription and promoter clearance
F
E
B
TFIID
H
initiation
TBP
J
1
RNA pol II
CTD
P
P
P

• RNA pol II is phosphorylated by TFIIH on the
  carboxy terminal
• domain (CTD), releasing it from the
  preinitiation complex and
• allowing it to initiate RNA synthesis and move
  down the gene

16
Transcription factors (partial list) Factor
Full name or function
CREB Cyclic AMP response
element binding protein CTF CAAT box
transcription factor (NF1) (binds
GGCCAATCT) NF1 Nuclear factor-1
(CTF) AP1 Activator protein-1 (dimer of the
Fos-Jun proteins) Sp1 Specificity factor-1
(binds CCGCCC) OTF Octamer transcription factor
(binds ATTTGCAT) NF-kB Nuclear factor
kB HSTF Heat shock transcription
factor MTF Metal transcription
factor USF Upstream factor ATF Activating
transcription factor HNF4 Hepatocyte nuclear
factor-4 (nuclear receptor superfamily) GR Glucoc
orticoid receptor (nuclear receptor
superfamily) AR Androgen receptor (nuclear
receptor superfamily) ER Estrogen receptor
(nuclear receptor superfamily) TR Thyroid
hormone receptor (nuclear receptor
superfamily) C/EBP CAAT/enhancer binding
protein E2F E2 factor (named for the adenovirus
E2 gene) p53 p53 (tumor suppressor
protein) Myc Product of the c-myc protooncogene
(dimerizes with Max)
17
Transcription factors (partial list) Factor
Full name or function
CREB Cyclic AMP response
element binding protein CTF CAAT box
transcription factor (NF1) (binds
GGCCAATCT) NF1 Nuclear factor-1
(CTF) AP1 Activator protein-1 (dimer of the
Fos-Jun proteins) Sp1 Specificity factor-1
(binds CCGCCC) OTF Octamer transcription factor
(binds ATTTGCAT) NF-kB Nuclear factor
kB HSTF Heat shock transcription
factor MTF Metal transcription
factor USF Upstream factor ATF Activating
transcription factor HNF4 Hepatocyte nuclear
factor-4 (nuclear receptor superfamily) GR Glucoc
orticoid receptor (nuclear receptor
superfamily) AR Androgen receptor (nuclear
receptor superfamily) ER Estrogen receptor
(nuclear receptor superfamily) TR Thyroid
hormone receptor (nuclear receptor
superfamily) C/EBP CAAT/enhancer binding
protein E2F E2 factor (named for the adenovirus
E2 gene) p53 p53 (tumor suppressor
protein) Myc Product of the c-myc protooncogene
(dimerizes with Max)
18

• Basic region-leucine zipper (bZIP) transcription
  factors
• Leucine zipper functions in dimerization
• Basic region binds DNA within the major groove
• Example of a bZIP transcription factor
• AP1 (Fos-Jun or Jun-Jun dimers)
• The Fos and Jun families each contain several
  different
• proteins that can homo- or heterodimerize
• Fos and Jun are products of the fos and jun
  protooncogenes
• AP1 is involved in the regulation of gene
  expression as
• controlled by various growth factors, hormones,
  tumor
• promoters, neuronal stimulation, and cellular
  stress

19
The Fos and Jun proteins
Fos
Jun
Basic regions (DNA contact surfaces
that bind to the DNA)

• Four leucines ( ) are present
• at every seventh position in
• the amphipathic a-helix

Leucine zipper (dimerization domain)
20
Helical wheel analysis of a leucine half-zipper
Leucine at every seventh position
Amphipathic alpha helix
21
Dimerization of the AP1 transcription factor
N
N
DNA binding domains

• Dimerization via the leucine
• zippers brings together the
• DNA binding domains of the
• two proteins, providing a
• sufficient amount of binding
• surface to form a stable
• protein-DNA interaction

• The leucine zippers interact via their
• hydrophobic faces forming coiled coils
• that cause the two proteins to dimerize

dimerized leucine zippers
C
C
22
Gcn4 (Basic Region, Leucine Zipper) Complex With
Ap-1 DNA Structures generated using RasWin
Molecular GraphicsWindows Version 2.6 and PDB
ID 1YSA
DNA binding
Leucine zipper
23
Binding of AP1 to DNA transactivates transcription
F
E
TFIID
B
H
TGACTCA ACTGAGT
TBP
A
J
1
RNA pol II

• Binding of AP1 to its
• DNA transcription element (TGACTCA)
  stimulates
• RNA synthesis by interacting with the
  preinitiation complex

24
Binding of AP1 to DNA transactivates transcription
F
E
P
TFIID
B
H
TGACTCA ACTGAGT
TBP
A
J
1
RNA pol II

• Binding of AP1 to its DNA transcription element
  (TGACTCA)
• Activity of AP1 can be further regulated by
  phosphorylation
• by Jun N-terminal kinase (JNK junk kinase)

25
Transcription factors (partial list) Factor
Full name or function
CREB Cyclic AMP response
element binding protein CTF CAAT box
transcription factor (NF1) (binds
GGCCAATCT) NF1 Nuclear factor-1
(CTF) AP1 Activator protein-1 (dimer of the
Fos-Jun proteins) Sp1 Specificity factor-1
(binds CCGCCC) OTF Octamer transcription factor
(binds ATTTGCAT) NF-kB Nuclear factor
kB HSTF Heat shock transcription
factor MTF Metal transcription
factor USF Upstream factor ATF Activating
transcription factor HNF4 Hepatocyte nuclear
factor-4 (nuclear receptor superfamily) GR Glucoc
orticoid receptor (nuclear receptor
superfamily) AR Androgen receptor (nuclear
receptor superfamily) ER Estrogen receptor
(nuclear receptor superfamily) TR Thyroid
hormone receptor (nuclear receptor
superfamily) C/EBP CAAT/enhancer binding
protein E2F E2 factor (named for the adenovirus
E2 gene) p53 p53 (tumor suppressor
protein) Myc Product of the c-myc protooncogene
(dimerizes with Max)
26
Zinc finger transcription factors
A C2H2 zinc finger
Cys
His
His
Cys
Cys
His
His
Cys

• each zinc finger consists of antiparallel
  b-sheets and an a-helix
• there are approximately 30 amino acid residues
  per finger domain
• a zinc atom is bound to two cysteine and two
  histidine residues (in C2H2)
• zinc finger proteins can have from 2 to over 30
  zinc finger domains
• zinc fingers of transcription factors bind to
  the major groove of DNA
• examples of zinc finger transcription factors
  include Sp1 and the
• steroid hormone receptors (nuclear receptor
  superfamily)
• some zinc fingers do not contain histidine
  (e.g., C4 and C5 zinc fingers)

27
The estrogen receptor
DNA binding domain
hormone binding, dimerization and
transactivation
C4 C5
transactivation
N
C
A C4 C5 zinc finger pair
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
Cys
28
Model for binding of steroid receptor dimer to DNA
one steroid receptor monomer (with two zinc
fingers)
the other steroid receptor monomer (with two zinc
fingers)
29
Binding of the estrogen receptor (ER) to DNA

• two subunits of an estrogen
• receptor dimer are shown
• bound to DNA
• each subunit has one of its
• two zinc fingers nestled into
• the major groove of the DNA
• the amino acid side chains of
• the zinc fingers recognize the
• DNA bases in dsDNA in a
• sequence-specific fashion

A G G T C A N N N T G A C C T T C C A G T N N N A
C T G G A
5-AGGTCANNNTGACCT-3 3-TCCAGT
NNNACTGGA-5
Estrogen response element (ERE)
30
Steroid hormone action in target cells
mifepristone (RU486) is a progesterone receptor
antagonist
31
Mutations affecting promoters

• The factor IX gene
• located on the X chromosome
• transcribed region gt32,700 bp, with 8 exons

• The factor IX gene promoter
• there are overlapping binding sites for AR and
  HNF4
• AR androgen receptor
• zinc finger nuclear receptor superfamily
  transcription factor
• binds androgen
• androgen levels increase at puberty
• HNF4 hepatocyte nuclear factor-4
• zinc finger nuclear receptor superfamily
  transcription factor
• ligand unknown - therefore an orphan receptor
• HNF4 is expressed early in development and in
  adult liver

-27
-15
-36
-22
HNF4
AR
32

• mutation at -20 results in
• Hemophilia B Leyden in which
• the hemophilia improves at puberty
• when levels of androgen increase

-27
-15
-36
-22
HNF4
AR

• mutation at -26 results in
• Hemophilia B Brandenburg
• in which factor IX levels remain low even after
  puberty