Basic transcription
mechanisms II
Thomas Dickmeis
Institut für Toxikologie und Genetik,
KIT, Karlsruhe
thomas.dickmeis@kit.edu
1
Moodle and exam
• Moodle: talks will be uploaded soon
– Password this year: IntroGen13
• Exam:
– Nick will set up a poll after Christmas to fix the date of
the exam
– Please send an email with your name and
Matrikelnummer to
nicholas.foulkes@kit.edu
2
Eukaryotic Transcription
3
Transcription in prokaryotes vs. eukaryotes
4
Stryer 2002
three important differences
• Chromatin is the template (bacteria: „naked“ DNA)
• Polymerase needs general transcription factors
(GTFs) for promoter binding and initiation
(bacteria: holoenzyme binds directly)
•
three polymerases (bacteria: one):
– RNA pol I: 18S/28S rRNA
– RNA pol II: mRNA, few small RNAs
– RNA pol III: tRNA, 5S rRNA, other small RNAs
5
Transcription in prokaryotes vs. eukaryotes
eubacteria
Nucleus
archaebacteria
eukaryotes
-
-
+
Transcription and
translation
not separated
not separated
separated
Genome organisation
one circular
chromosome
one circular
chromosome
several linear
chromosomes
-
-
+
few
few
majority
operons
+
+
- (exceptions?)
Introns
-
-
+
RNA polymerases
1
1
3:
Pol I = rRNA
Pol II= mRNA
Pol III = tRNA
eubacterial
archaebacterial
archaebacterial
5´Cap on mRNA
-
-
+
polyA 3´ on mRNA
-
rare
+
Histones/
nucleosomes
Non-coding sequences
RNA polymerase type
6
Typical regulatory sequences of a Pol II
transcribed gene
Promoter:
- binds GTFs
Enhancer:
- binds transcriptional regulators
- increases promoter utilization
- can be upstream, inside the gene or downstream
(„distal“ enhancers can be very far away)
- orientation not important
- often target for tissue-specific or temporal regulation
Silencer:
same, but decreases promoter utilization
(How can distant sequences influence the promoter?)
7
DNA looping brings enhancers and
promoters together
(How can one prove this?)
8
Cooper 2000
The 3C technique allows the study of
chromatin looping
„3C“ = Chromosome Conformation Capture
(How can one
avoid
intermolecular
ligation?)
(What has to be
known in this
case?)
9
Chromatin and transcription
10
Alberts 2002
Principles of enhancer function:
I. making the promoter accessible
ATP
ADP+P
1. Chromatin remodelling
makes the promoter accessible
(How does one know if an octamer
has been displaced?)
11
(finding the accessible chromatin:
mapping of DNAseI hypersensitive sites)
12
Principles of enhancer function:
I. making the promoter accessible
2. Chromatin modifications
Generation of chromatin marks
that can be bound e.g. by the
basal transcription factors
13
Principles of enhancer function:
II. Architectural proteins
Bending of the DNA to facilitate or prevent interaction of other factors
14
Principles of enhancer function:
III. Interaction with the basal transcription apparatus
Activators may directly
interact with the basal
transcription apparatus;
often via a special domain
- the activation domain
(AD)
Activators can also interact
indirectly via separate
factors, so called
coactivators
The same is true for repressors:
Direct interaction via repressor domains or interaction via corepressors
15
The mediator complex links transcriptional
regulators with the basal transcription apparatus
Pol II
Mediator is required for transcription from most Pol II dependent promoters in yeast –
sometimes referred to as being a GTF itself
16
Alberts 2002
Nature Structural & Molecular Biology 11, 394 - 403 (2004)
The mediator complex links transcriptional
regulators with the basal transcription apparatus
The modular structure of mediator allows
interaction with different transcription
factors, coactivators and components of the
basal transcription apparatus
(Cartoon! Not all interactions present at the
same promoter....)
17
Nature Structural & Molecular Biology 11, 394 - 403 (2004)
Current Opinion in Genetics & Development 18:397–403 (2008)
Integration at promoters
Promoters can function as genetic switches that integrate regulatory information
MODULARITY of regulatory input is a recurring theme
18
Alberts 2002
Summary eukaryotic
transcriptional regulation principles
1.
Enhancer:
activating regulatory sequence separate from core promoter
– independent from distance and orientation
2. Enhancers bind activating transcription regulators
(repressing factors bind to silencers)
3. Enhancers may function
– in making the promoter accessible
(chromatin remodelling and modifications)
– changing DNA topology (e.g.bending)
– interacting with the basal transcription apparatus
4. Promoters integrate information from various regulatory
elements (modularity)
More about all this in Clemens Grabher‘s lecture
19
RNA pol II promoter
Nature. 2009 Sep 10;461(7261):186-92
Modular: can contain e.g.
Inr – Initiator region
TATA box
or
DPE – Downstream Promoter Element
(in TATA-less promoters)
TATAA
YYCAYYYY
AGAC
reminiscent of
prokaryotes:
TTGACA
TATAAT
20
The core promoter is bound by general
(or „basal“) transcription factors (GTFs)
A promoter recognition factor binds the promoter:
TFIID (Transcription Factor for RNA pol II D)
Consistst of many subunits:
TBP – TATA Binding Protein
TAFs – TBP Associated Factors
idealized cartoon:
subunit composition varies a lot –
different TFIIDs recognize different
promoters
21
Nat Rev Genet. 2010 Aug;11(8):549-58
TBP bends DNA at the TATA box
Widens the minor
groove
Brings proteins
binding to the
promoter into closer
proximity
In some complexes,
TBP is present but
does not bind DNA
22
The different core promoter types are bound
by different promoter recognition factors
Müller F et al. J. Biol. Chem. 2007;282:14685-14689
Differential expression of core
promoter recognition factors may
contribute to cell type specific
transcription regulation
CpG islands –
a hallmark of „housekeeping genes“23
CpG islands
• found in housekeeping genes: constitutively expressed
genes
• increased density of the dinucleotide CG at the 5‘ end
• CpGs less frequent in the rest of the genome – the Cs
get methylated by DNA–methyl-transferases – then
frequently disappear – why?
methylation
spontaneous
deamination
• Mutation to T
24
Stryer 2002
CpG islands
In active promoters, DNA
should be demethylated
Promoters active in the
germline are spared of
methylation
-> less mutation of C to T
25
Alberts 2002
(How can methylation be detected?)
Some restriction enzymes are sensitive to methylation of their recognition sites
26
Summary eukaryotic promoters
1.
2.
3.
4.
5.
6.
Modular (as in prokaryotes)
Frequent motifs: TATA, Inr, DPE
Various classes of promoters combine different motifs
Promoter recognition complexes bind the promoters
Classic example: TFIID (TBP and TAFs)
Different recognition complexes binding different
promoter classes may contribute to cell type specific
regulation of transcription
7. CpG islands are a feature of housekeeping genes and
reflect the demethylated state of their promoter DNA
27
Assembly of the basal transcription
apparatus
=
After the
binding of
TFIID, other
TFIIs and the
polymerase
itself bind:
initiation
complex
transforms into
elongation
complex
28
First assembly steps
TFIIA:
- TFDII can bind to region extending farther upstream
TFIIB:
- binds adjacent to TBP (BRE - B Recognition Element)
- determines promoter polarity
- recruits the polymerase
TFIIF:
- binds polymerase
- facilitates recruitment
29
Eukaryotic RNA polymerases
Cartoon of protein gel
from yeast RNA polymerase II:
The bacterial subunits:
especially catalytic units conserved
no sigma – role fulfilled by the GTFs
enzyme alone can transcribe, but not initiate
30
(archaeal and eukaryotic RNA polymerases
are related)
31
Trends in Microbiology 6/6, 222-228 (1998)
Open complex formation and
promoter clearance
TFIIE:
- facilitates formation of initiation-competent polymerase
- recruits TFIIH
TFIIH:
- multiple enzymatic activities
- helicase -> melting of the DNA
CTD-domain of the RNA Pol II gets phosphorylated – leaves promoter and
starts to elongate
32
Phosphorylation of proteins is an effcient
way of regulation
The reaction is catalysed by protein kinases, which are target selective
Phosphorylation may :
- cause conformational changes
- create or abolish binding sites for other proteins
Phosphate groups may be removed by selective phosphatases
33
Stryer 2002
Phosphorylation of the CTD regulates
transcription
The heptad repeat:
(YSPTSPS)n=26-52
Ser2 Ser5
Mediator binds unphosporylated CTD
9, 810-815 (October 2008)
Alberts 2002
CTD
TFIIH phosphorylates Ser5 -> promoter clearance
P-TEFb phosphorylates Ser2 -> escape from pausing
34
Stalled transcription –promoter-proximal
pausing
Polymerase on Drosophila heat shock genes stalled 50 bp downstream of the TSS
Released by P-TEFb:
-> phosphorylates pausing associated factors and the CTD-Ser2
-> proper elongation
35
Chromosoma (2009) 118:1–10
Regulation by transcription factors at
different steps may save different purposes
Pho4: chromatin opening
HSF: escape from pausing
P-TEFb
• tight control via promoter accessibility
and subsequent initiation steps
• can be relatively slow
(example: acid phosphatase gene should only
be induced when the cell needs phosphate)
• rapid activation of paused polymerase
• control may be leaky
(example: heat shock genes need to respond
rapidly to heat stress)
Nature. 2009 Sep 10;461(7261):186-92
HSF
The CTD code
A CTD code for different phases of transcription
37
Nat.Rev.Gen.10:457-466 (2009)
CTD code and integration with RNA
processing
the „cap“
Stryer 2002
2008, 20:260–265
More about RNA modifications in Harald König‘s lecture
38
Semi Cell & Dev Biol 18 (2007) 691–697
The „transcription factory“ model
a cell nucleus
stained for
phosphylated
Pol II (red)
Polymerases are localized
and thread DNA through the
„factory“
Nat Rev Genet. 2009 Jul;10(7):457-66.
Such factories can also be associated
with zones enriched for splicing factors
39
(„nuclear speckles“)
Transcription and DNA repair
Transcription and genome
integrity affect each other, e.g.
DNA lesions inhibit progress of
the polymerase -> repair
TFIIH participates in both
processes
(Human disease genes:
Xeroderma pigmentosum: e.g. XPB, XPD
Cockayne syndrome: CSA, CSB)
Also:
Transcription can affect
mutagenesis or
recombination rates
More on DNA repair in Felix Loosli‘s lecture
40
Summary pol II transcription
1. The initiation complex assembles at the site of core
promoter recognition factor binding
2. The TFIIH helicase function assists in promoter
melting
3. The TFIIH kinase function phosphorylates the CTD
domain of the polymerase (Ser5) – promoter escape
4. Many genes have paused polymerases near their 5‘
end
5. PTEFb kinase phosphorylates the CTD (Ser2) –
productive elongation
6. CTD is differentially phosphorylated throughout the
transcription cycle – the CTD code
7. Transcription and RNA processing are integrated –
mediated by CTD code
8. Transcription factories - spatial organization of
transcription in the nucleus
9. Transcription and DNA repair are linked (TFIIH)
41
Cell 133, May 16, 2008
(Sequential vs. holoenzyme assembly)
42
Critical Reviews in Biochemistry and Molecular Biology, 41:105–178, 2006
5 minutes break !
43
The other polymerases
Ribosomal subunits are assembled in the nucleolus
Ribosomes consist of
proteins and RNA
(more in Felix and
Clemens‘ lectures!)
Alberts 2002
Which polymerases are required for ribosome synthesis?
Pol I
Pol III
Pol II
Pol I and III can constitute up to 80% of all transcription in rapidly growing cells!
44
Cooper 2002
The other polymerases: Pol I
„christmas tree“ transcription
of tandem rDNA arrays
Alberts 2002
Synergistic binding of UBF and SL1
to the promoter recruits PolI and
associated factors
45
The other polymerases: Pol I
Many genes involved in
cancer regulate
components of the Pol I
machinery –
Reflects the importance
of ribosome synthesis
for cell growth and
proliferation
46
The other polymerases: Pol III
Pol III transcribes a whole battery of small RNAs,
the most abundant of which are tRNAs and 5S rRNA
Dec;23(12):614-22 (2007)
47
The other polymerases: RNA pol III
Dec;23(12):614-22 (2007)
Basal RNA pol III promoter elements can be downstream of the transcription start site
48
The other polymerases
All three classes of polymerases
bind via commitment factors
TBP is involved in initiation in all
three classes
(not always binding to the DNA)
49
The other polymerases
organelles
Human mitochondrial RNA polymerase
Molecular Cell 24, 813–825, 2006
Bakteriophage T7 RNA polymerase
(In chloroplasts more complicated:
NEP – nuclear encoded, phage type
PEP – plastid encoded, eubacterial type)
Single subunit polymerases
Alberts 2002
50
(T7 often used in the lab for in vitro transcription)
Other polymerases: summary
• 3 eukaryotic polymerases:
- RNApol I: rRNA (18S, 28S)
- RNA pol II: mRNAs, other small RNAs
- RNA pol III: tRNAs, 5S rRNA, other small RNAs
(+ organelle polymerases:
- in mitochondria: phage-like
- in chloroplasts: both phage and eubacterial type)
• TBP required for promoter recruitment in all 3
51
Some examples of current
research
52
Quantifying transcription of the genome:
RNA seq
http://en.wikipedia.org/wiki/File:RNA-Seq-alignment.png
53
Nature Methods Vol5 No7 ,JULY 2008 pp.621-28
Global mapping of transcription start sites
CAGE technology: „Cap Analysis of Gene Expression“
54
http://www.riken.go.jp/engn/r-world/research/lab/osc/genotech/images/01b.gif
Different types of promoters detected with
CAGE
55
Nat Rev Genet. 2007 Jun;8(6):424-36
Different types of promoters
May be linked with different
core promoter recognition factors
Müller F et al. J. Biol. Chem. 2007;282:14685-14689
56
Large scale sequencing of transcripts
• Mapping of start sites
• Alternative transcripts
• Other transcripts, also many from intergenic regions
• „Pervasive transcription“ of the genome
„Dark
matter transcripts“
57
„Dark matter“ transcripts?
Based on RNA seq results as opposed to the tiling array method
=> When a new technology is introduced, be aware of artefacts!
58
„Dark matter“ transcripts?
However, the story continues….:
Many newly discovered types of transcripts
are associated with promoters
Just transcriptional „noise“?
Or specific functions?
60
Nat Rev Genet. 2009 Dec;10(12):833-44
Transcription at enhancers?
eRNAs =
enhancer RNAS
When the promoter is missing, RNA polymerase still
sits at the enhancer, but no transcription occurs
Function of all this?
61
Thanks for your attention!
62
References
Pictures without reference are from Lewin‘s Genes X, © Jones and
Barlett publishers, LLC (www.jbpub.com)
Pictures with the following reference are from:
Alberts 2002: Alberts, Molecular Biology of the Cell:
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4
Stryer 2002: Stryer, Biochemistry:
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer
Cooper 2002: Cooper, The Cell – A Molecular Approach:
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper
Knippers 1997: Rolf Knippers, Molekulare Genetik, 7. Auflage, Thieme
1997
63