Transcript Transcription Eukary 2

Gene Expression
Eukaryotic Gene Transcription
9/18/08
Thomas Ryan, Ph.D.
Biochemistry and Molecular Genetics
[email protected]
Eukaryotic RNA Polymerases
• Three DNA dependent RNA polymerases:
RNA Pol I, II, and III
• All 3 are big, multimeric proteins (500-700 kD)
• All have 2 large subunits with sequences similar to 
and ' in E. coli RNA polymerase, so catalytic site
may be conserved
• All interact with general transcription factors-GTFs
• RNA Pol II is most sensitive to -amanitin
RNA Polymerase II Inhibitor -Amanitin
“The Destroying Angel” - Amanita phalloides
pol II >> pol III >>> pol I
Bicyclic octapeptide
Blocks elongation
Yeast RNA Polymerase II Subunits
Transcription Factors

The three RNA polymerases (I, II and III) interact with their
promoters via protein:protein and protein:DNA interactions

These proteins or transcription factors (TFs) recognize and
initiate transcription at specific promoter sequences

Some transcription factors (TFIIIA and TFIIIC for RNA
polymerase III) bind to specific recognition sequences within
the coding region
Helix-Turn-Helix Motif
Zinc-Finger Motif: C2H2 Class
Basic Region-Leucine Zipper Motif: bZIP
bZIP Transcription Factor
General Transcription Factors (GTFs)
• GTFs position RNAPs at transcription initiation sites,
forming the preinitiation complex (PIC)
• Transcription-initiation complex = RNAP + general
transcription factors (GTF) bound to promoter region
• Many of the GTFs that associate with RNAP II initiate
transcription from TATA box-containing promoters have
been identified
– TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
– TFII = “transcription factor RNAP II”
General Transcription Factors
• TFIID is largest and consists of a TATA-box binding protein
(TBP) and 8-10 TBP-associated factors (TAFIIs)
• TBP is a “universal transcription factor” – associates with
promoters of all three RNAPs, and promoters with and
without a TATA box
• Binding of TAFIIs extend the interactions of TFIID
• TFIID has two roles:
– foundation for the transcriptional PIC complex
– Prevents nucleosome stabilization in the promoter
region (antagonist to H1)
TBP is used by all 3 RNA polymerases
• TBP is a subunit of an important GTF for each of the 3 RNA
polymerases:
– TBP or TFIID for Pol II
– SL1 for Pol I
– TFIIIB for Pol III
• It does NOT always bind to TATA boxes; promoters for RNA Pol I and
Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still
used.
• The GTFs that contain TBP may serve as positioning factors for their
respective polymerases.
Yeast TATA Binding Protein: TBP
TBP binds in the minor groove of DNA
TBP binding bends the DNA
Promoters for RNAP I
• RNA Pol I transcribes genes for the large rRNA precursor
• There are hundreds of similar copies of this gene in each genome
• RNA Pol I promoters (called class I) have two components:
• Upstream control element: –156 to -107
• Core element: –45 to +20
• Two different transcription factors bind these sequences cooperatively: SL1 and UBF (TBP is a component of SL1)
Promoters for RNAP III

Transcribes various small RNAs: 5S rRNA, tRNA
precursors, U6 snRNA, etc


The 5S rRNA and tRNA genes have class III promoters

The 5S rRNA promoters are entirely within the coding
region of the gene
U6 snRNA and 7SL RNA gene have promoters that
resemble RNAP II promoters
 The tRNA promoters contain two elements
 The other promoters for U6 snRNA etc appear similar to
RNAP II promoters (have TATA boxes) but -amanitin test
indicates these are transcribed by RNAP III.
PIC Assembly for RNA Pol III Genes
TBP
TBP
TBP
TBP
RNA Polymerase II
General Transcription Factors
Factor
Subunits Function
TFIID - TBP
TFIID - TAFs
1
12
TFIIA
TFIIB
TFIIF
RNA Pol II
TFIIE
3
1
2
12
2
TFIIH
9
Recognize core promoter (TATA), Recruit TFIIB
Recognize core promoter (non-TATA), Pos and Neg
regulatory functions, HAT activity
Stabilize TFIID and promoter binding
Recruit RNA Pol II / TFIIF, Start site selection
Assist RNA Pol II to bind promoter
Enzymatic synthesis of RNA, Recruit TFIIE
Recruit TFIIH, Modulate TFIIH helicase, ATPase,
and kinase activities
Promoter melting using helicase, Promoter
clearance via CTD phosphorylation
Modified from Roeder, R.G., Trends in Biochem. Sci. 21:327-334
TBP-associated Factors (TAFs or TAFIIs)
• Important for helping TBP to bind to promoters that
lack TATA boxes.
• There are different TAFs in different cells!
• In vivo these factors are associated with additional
proteins forming a larger complex of about 50
polypeptides.
• It is hypothesized that this high MW complex may
preassemble and interact with promoters in a single step.
Eukaryotic RNA Pol II Transcription
Formation of the PIC

TFIID binds TATA
box via TBP subunit
 TFIIA facilitates
and stabilizes
binding of TFIID
complex
Eukaryotic RNA Pol II Transcription
Formation of the PIC
TFIIB binds to TFIID
TFIIB is a monomeric protein

C terminal domain contacts
DNA and TBP

N terminal domain extends
towards start site

Proximity of certain promoterand enhancer-transcription
factors (important for
developmental regulationtheir activation domain binds
directly to TFIIB)

TFIIH close as well at this
point, but not yet bound
Eukaryotic RNA Pol II Transcription
Formation of the PIC
 TFIIF binds to RNAPII (preformed
complex) – directs RNAPII to
promoter
 Binding of TFIIE to TFIIF/RNAPII
complex and already positioned
TFIIB helps positioning the
RNAPII over start site:
 Two large subunits of RNAP II
interact with promoter DNA: CTD
tail (unphsphorylated form) of
RNAPII is in direct contact with
TFIID
 TFIIE is DNA-dependent ATPaseprobably necessary for
generating the energy for
transcription
Eukaryotic RNA Pol II Transcription
Formation of the PIC
Binding of RNAP II/TFIIF/TFIIE to
promoter activates TFIIH
 TFIIH contains nine subunits
 It has helicase activity- unwinds
DNA downstream from the
initiator site in the presence of
ATP necessary for promoter
clearance
 It has protein kinase activityphosphorylation of CTD tail of
RNAPII
 Phosphorylation detaches
RNAPII from TFIID
 Beginning of transcription by
RNAPII
Carboxyl-Terminal Domain (CTD Tail)
• Stretch of 7 amino acids that is
repeated multiple times (26-52
times):
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
• Critical for viability
• CTD tail becomes phosphorylated on
ser and some tyr residues as the
RNAP transcribes away from the
promoter
RNA Pol II Promoters
Consist of two parts:
1. Core promoter:
- TATA box (position at ~ -30)
- initiator (on the transcription start site)
2. Proximal Promoter Elements (can be upstream,
downstream or internal)
Upstream
Element
TATA
Initiator
Downstream
Element
Eukaryotic TATA Box
TATA motif is usually located at position -25
Consensus
Sequence
Eukaryotic Promoter Regions
Initiator
RNA Polymerase II Promoter Consensus Sequences
of Transcription Factor Binding Sites
Proximal Control Elements of Genes
Modular Factor Binding Sites
Coordinate Regulation Via Response Elements
 Multiple genes are transcribed in response to different cues: for
example, heat shock, hormone levels, developmental events,
phorbol esters, heavy metals, metabolite concentrations, etc.
 Similarly responsive genes will have a DNA sequence located in
cis to the gene called a response element.
 These response elements are binding sites for transacting
factors that are activated in response to the environmental cue.
 The location of these elements relative to the start site of
transcription is not conserved between genes: eg. a cis element
that leads to transcription in response to a hormone may be
located at –300 in one gene and –175 in another
Response Elements: Coordinate Regulation
Metallothionein Gene Promoter
Coordinate Regulation by Hormones/Steroid Receptors
Adapted from Molecular Biology of the Cell, 4th Edition
Steroid
Receptors
Upon binding to the hormone
cortisol,
the
cytoplasmic
glucocorticoid receptor displaces
an inhibitory protein (Hsp90)
and moves to the nucleus where
is
can
interact
with
glucocorticoid response elements
(GREs) in the DNA affecting
gene transcription.
Adapted from Molecular Biology of the Cell, 4th Edition
Activation of Transacting Factors
Control of Cellular Differentiation By TFs
Enhancers
 Control elements that stimulate transcription
 Bind multiple different transcription factors
 Transcription factors that recognize enhancer =
activators or enhancer binding proteins
 Activators interact with general transcription factors
 Negative enhancer is a silencer
Enhancers
Stimulate expression of genes over long
disances (up to 50kb)
Occur upstream, downstream, in introns
or in exons
Orientation independent
May be cell-type specific
Enhancers: Action at a Distance
Activation of transcription
initiation in eukaryotes by
recruitment of the
eukaryotic RNA polymerase
II holoenzyme complex (100
protein subunits).
Insulators / Boundary Elements
Boundary elements block encroachment of heterochromatin from
neighboring loci. They also stop the unregulated enhancement or
activation of neighboring genes outside of their chromosomal
domain.
Hypomethylation
(Active)
Vs.
Hypermethylation
(Silenced)
Silencing: Histone Deacetylation HDAC
• Some repressors recruit histone deacetylase, which removes
acetyl groups from histones resulting in gene silencing
Histone Acetylation and Deacetylation
+
NH 3
CH2
CH2
O
CH2 O
CH2
... NH CH C NH CH C ...
2
Gly
Lys
Positive charge on amino group
Histone acetyl
transferases
HAT
AcCoA
CH3
O C
NH
CH2
CH2
CoA
O
CH2 O
CH2
... NH CH C NH CH C ...
2
HDAC
Histone
deacetylases
Ac
No charge on amide group
Histone Code
Histone tails are
postranslationally
modified by
acetylation,
methylation,
phosphorylation and
ubiquitination. These
modifications have a
profound effect on
gene activity. The
specific set of
modifications is
termed the “histone
code” .
Chromatin Remodeling
Remodeling complexes
allow access of replication
and transcription factors to
the DNA.
Remodeling requires ATP
Gene Activation By Chromatin Remodeling
Transcription
Factor Access
HATs
open chromatin
Nucleosome
remodelers
Transcription Factor
Coactivators
Assembly of
preinitiation
complex on
open chromatin
Chromatin remodeling of diploid somatic cell
nucleus in the egg cytoplasm reprograms the
nucleus to recapitulate development.