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Transcript of Basic transcription mechanisms II Thomas Dickmeis Institut für Toxikologie und Genetik, KIT,...
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Basic transcription mechanisms II
Thomas Dickmeis
Institut für Toxikologie und Genetik,
KIT, Karlsruhe
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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
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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
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Transcription in prokaryotes vs. eukaryoteseubacteria archaebacteria eukaryotes
Nucleus - - +
Transcription and translation
not separated not separated separated
Genome organisation one circular chromosome
one circular chromosome
several linear chromosomes
Histones/nucleosomes
- - +
Non-coding sequences few few majority
operons + + - (exceptions?)
Introns - - +
RNA polymerases 1 1 3:Pol I = rRNAPol II= mRNAPol III = tRNA
RNA polymerase type eubacterial archaebacterial archaebacterial
5´Cap on mRNA - - +
polyA 3´ on mRNA - rare +
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Typical regulatory sequences of a Pol II transcribed gene
(How can distant sequences influence the promoter?)
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
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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?)
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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?)
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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
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Principles of enhancer function:II. Architectural proteins
Bending of the DNA to facilitate or prevent interaction of other factors
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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
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The mediator complex links transcriptional regulators with the basal transcription apparatus
Alberts 2002 Nature Structural & Molecular Biology 11, 394 - 403 (2004)
Pol II
Mediator is required for transcription from most Pol II dependent promoters in yeast – sometimes referred to as being a GTF itself
17Nature Structural & Molecular Biology 11, 394 - 403 (2004)
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....)
The mediator complex links transcriptional regulators with the basal transcription apparatus
Current Opinion in Genetics & Development 18:397–403 (2008)
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Integration at promoters
Promoters can function as genetic switches that integrate regulatory information
MODULARITY of regulatory input is a recurring theme
Alberts 2002
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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
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RNA pol II promoter
TATAATTTGACA
Nature. 2009 Sep 10;461(7261):186-92
TATAA YYCAYYYY AGAC
Modular: can contain e.g.Inr – Initiator regionTATA boxorDPE – Downstream Promoter Element(in TATA-less promoters)
reminiscent of prokaryotes:
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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
Nat Rev Genet. 2010 Aug;11(8):549-58
idealized cartoon:subunit composition varies a lot – different TFIIDs recognize different promoters
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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
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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“
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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?
• Mutation to TStryer 2002
spontaneous
deamination
methylation
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CpG islands
In active promoters, DNA should be demethylated
Alberts 2002
Promoters active in the germline are spared of methylation-> less mutation of C to T
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(How can methylation be detected?)
Some restriction enzymes are sensitive to methylation of their recognition sites
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Summary eukaryotic promoters
1. Modular (as in prokaryotes)
2. Frequent motifs: TATA, Inr, DPE
3. Various classes of promoters combine different motifs
4. Promoter recognition complexes bind the promoters
5. Classic example: TFIID (TBP and TAFs)
6. 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
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Assembly of the basal transcription apparatus
=After the binding of TFIID, other TFIIs and the polymerase itself bind:initiation complex
transforms into elongation complex
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First assembly stepsTFIIA: - 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
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Eukaryotic RNA polymerases
The bacterial subunits:
Cartoon of protein gel from yeast RNA polymerase II:
especially catalytic units conservedno sigma – role fulfilled by the GTFsenzyme alone can transcribe, but not initiate
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Open complex formation andpromoter 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
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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
Stryer 2002
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Alberts 2002
Phosphorylation of the CTD regulates transcription
9, 810-815 (October 2008)
(YSPTSPS)n=26-52
Ser2 Ser5
Mediator binds unphosporylated CTD
TFIIH phosphorylates Ser5 -> promoter clearance
P-TEFb phosphorylates Ser2 -> escape from pausing
The heptad repeat:
CTD
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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
Chromosoma (2009) 118:1–10
Regulation by transcription factors at different steps may save different purposes
Pho4: chromatin opening HSF: escape from pausing
• 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)
Nat
ure.
200
9 S
ep 1
0;46
1(72
61):
186-
92
• rapid activation of paused polymerase • control may be leaky (example: heat shock genes need to respond rapidly to heat stress)
HSF
P-TEFb
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CTD code and integration with RNA processing
More about RNA modifications in Harald König‘s lecture
2008, 20:260–265
Stryer 2002
the „cap“
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The „transcription factory“ model
Such factories can also be associated with zones enriched for splicing factors(„nuclear speckles“)
Polymerases are localized and thread DNA through the „factory“
Nat Rev Genet. 2009 Jul;10(7):457-66.
a cell nucleus stained for phosphylated Pol II (red)
Sem
i Cel
l & D
ev B
iol 1
8 (2
007)
691
–697
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Transcription and DNA repair
More on DNA repair in Felix Loosli‘s lecture
Transcription and genome integrity affect each other, e.g.
DNA lesions inhibit progress of the polymerase -> repair
TFIIH participates in both processes
Also:Transcription can affect mutagenesis or recombination rates
(Human disease genes:Xeroderma pigmentosum: e.g. XPB, XPDCockayne syndrome: CSA, CSB)
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Summary pol II transcription
Cell 133, May 16, 2008
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)
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(Sequential vs. holoenzyme assembly)
Critical Reviews in Biochemistry and Molecular Biology, 41:105–178, 2006
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The other polymerases
Alberts 2002
Ribosomes consist of proteins and RNA
Ribosomal subunits are assembled in the nucleolus
Cooper 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!
(more in Felix and Clemens‘ lectures!)
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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
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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
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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)
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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
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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)
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The other polymerases
Alberts 2002
Human mitochondrial RNA polymerase
Bakteriophage T7 RNA polymerase
Single subunit polymerases(T7 often used in the lab for in vitro transcription)
Molecular Cell 24, 813–825, 2006
(In chloroplasts more complicated:NEP – nuclear encoded, phage typePEP – plastid encoded, eubacterial type)
organelles
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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
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Quantifying transcription of the genome:RNA seq
Nature Methods Vol5 No7 ,JULY 2008 pp.621-28
http://en.wikipedia.org/wiki/File:RNA-Seq-alignment.png
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Global mapping of transcription start sitesCAGE technology: „Cap Analysis of Gene Expression“
http://www.riken.go.jp/engn/r-world/research/lab/osc/genotech/images/01b.gif
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Different types of promoters
Müller F et al. J. Biol. Chem. 2007;282:14685-14689
May be linked with different core promoter recognition factors
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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“
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„Dark matter“ transcripts?
=> When a new technology is introduced, be aware of artefacts!
Based on RNA seq results as opposed to the tiling array method
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Many newly discovered types of transcripts are associated with promoters
Nat Rev Genet. 2009 Dec;10(12):833-44
Just transcriptional „noise“?
Or specific functions?
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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?
63
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