Basic transcription mechanisms II Thomas Dickmeis Institut für Toxikologie und Genetik, KIT, Karlsruhe [email protected] 1.

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Basic transcription mechanisms II

Thomas Dickmeis

Institut für Toxikologie und Genetik,

KIT, Karlsruhe

[email protected]

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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

[email protected]

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Eukaryotic Transcription

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Transcription in prokaryotes vs. eukaryotes

Stryer 2002

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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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DNA looping brings enhancers and promoters together

Cooper 2000

(How can one prove this?)

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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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Chromatin and transcription

Alberts 2002

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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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(finding the accessible chromatin: mapping of DNAseI hypersensitive sites)

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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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(archaeal and eukaryotic RNA polymerases are related)

Trends in Microbiology 6/6, 222-228 (1998)

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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-

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• 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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The CTD code

Nat.Rev.Gen.10:457-466 (2009)

A CTD code for different phases of transcription

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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

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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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5 minutes break !

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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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Some examples of current research

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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 detected with CAGE

Nat Rev Genet. 2007 Jun;8(6):424-36

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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

„Dark matter“ transcripts?

However, the story continues….:

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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?

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Thanks for your attention!

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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