Zyklusvorlesung Molekularbiologie WS 2009/10
Victor Sourjik, Seite 1
Kontrolle der Genexpression
Victor Sourjik, ZMBH
Zyklusvorlesung Molekularbiologie WS 2009/10
Victor Sourjik, Seite 2
Wie wird das Expressionsniveau
reguliert?
Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008)
Zyklusvorlesung Molekularbiologie WS 2009/10
Victor Sourjik, Seite 3
Genexpression bei Bakterien und
Eukaryonten
Differences:
- Genome organisation
- Gene- / Transcript structure
- Processing of RNA
- RNA Degradation
- RNA Transport
- Translation
Evolutionary significance of these differences?
Figure 6-21 Molecular Biology of the Cell (© Garland Science 2008)
Zyklusvorlesung Molekularbiologie WS 2009/10
Victor Sourjik, Seite 4
Genexpressionskontrolle bei Eukaryonten
Figure 7-5 Molecular Biology of the Cell (© Garland Science 2008)
Eucaryotes have more opportunities to regulate gene expression than bacteria
Zyklusvorlesung Molekularbiologie WS 2009/10
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Vielfalt der RNA Moleküle in der Zelle
Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)
What RNA polymerases transcribe what RNAs?
Zyklusvorlesung Molekularbiologie WS 2009/10
RNA-Polymerasen
Bacteria have only one RNA polymerase
Eucaryotes have three RNA polymerases:
Table 6-2 Molecular Biology of the Cell (© Garland Science 2008)
Victor Sourjik, Seite 6
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Transkriptionskontrolle (bei Bakterien)
Initiation
Elongation
5‘ UTR
Promoter
Coding region
Termination
3‘ UTR
DNA
terminator
+1 (start site)
Transcription
5‘
3‘
RNA
AUG
UGA
Initiation of transcription is the main point of regulation
(both in bacteria and in eucaryotes)
Zyklusvorlesung Molekularbiologie WS 2009/10
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Transkriptionsinitiation (bei Bakterien)
binding




closed
complex
isomerisation
open
complex
initiation
synthesis
start
promoter
clearance
elongation
Assembly of the (pre)initiation complex (PIC) on promoter is the
most frequently regulated step in bacteria and in eucaryotes
Zyklusvorlesung Molekularbiologie WS 2009/10
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„Passive“ und „aktive“ Transkriptionsregulation
Passive regulation by promoter strength
Sigma 70 consensus promoter in E. coli:
TTGACA...17bp...TATAAT

TTCAAA...17bp...TAATAT



TTGAAA...17bp...TATAAT
Zyklusvorlesung Molekularbiologie WS 2009/10
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„Passive“ und „aktive“ Transkriptionsregulation
Active regulation
Bacteria:
-Sigma Factors
-Activators
-Repressors




Eucaryotes:
-Chromatin remodeling factors
-General transcription factors (GTFs)
-Activators
-Repressors
-Covalent Modifications
Pol II
GTFs
Zyklusvorlesung Molekularbiologie WS 2009/10
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Wirkungsmechanismen von
Transkriptionsfaktoren (Bakterien)
Activators
binding to 
subunit
binding to 
subunit
conformation
al change
in promoter
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Wirkungsmechanismen von
Transkriptionsfaktoren (Bakterien)
Repressors
steric
hindrance
DNA
looping
modulation
of
an activator
Zyklusvorlesung Molekularbiologie WS 2009/10
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Signalintegration (Bakterien)
repositioning
independent contacts
cooperative binding
anti-repression
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Promotor- und Genstruktur bei Bakterien
und Eukaryonten
-35
-10
5‘ TTGACA...17bp...TATAAT 3‘
Bacterial promoter
UP -35 -10
Binding sites for activators and
repressors
GC box (-90)
terminator
+1 (start site)
5‘ TATAA 3‘
Eucaryotic promoter
TATA box
(-30)
cleavage and poly-A signal
CAAT box (-75)
Inr (-3 to +5)
DPE (+30)
5‘ YYCAYYYYY 3‘ 5‘ AGAC 3‘
Proximal binding sites for activators and repressors; enhancers/UASs; silencers; insulators etc
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Allgemeine Transkriptionsfaktoren bei
Eukaryonten
Figure 6-16 Molecular Biology of the Cell (© Garland Science 2008)
Figure 6-17 Molecular Biology of the Cell (© Garland Science 2008)
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Initiationskomplex und Aktivatoren
Eucaryotic initiation complex
consists of multiple general and
specific TFs ->
Multiple opportunities of
regulation
-Similar to bacterial activators, many
eucaryotic activators assist assembly of
(pre)initiation complex
-Other activators recruit histone
modifiers and chromatin remodelers
-Mediator complex offers multiple binding
sites for PIC assembly
Figure 7-44 Molecular Biology of the Cell (© Garland Science 2008)
-Eucaryotic TFs can bind to the regulatory
sequences (enhancers) far upstream or
downstream from the transcription start site
(TSS)
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Wichtiges Konzept: DNA looping and
persistence length
Figure 7-41 Molecular Biology of the Cell (© Garland Science 2008)
DNA is stiff at short distances but flexible at long distances
(persistence length von ca. 200 bp)
=> Binding sites that are farther apart can easier come close to each other!
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Chromatinstruktur und Transkriptionsinitiation
Pol II
GTFs
Eucaryotic promoter DNA must be freed or at least loosened from nucleosomes to
allow assembly of the initiation complex:
Nucleosomes hinder TF binding to the DNA
DNA sequence influences nucleosome positioning
„Pioneer“ TFs bind at nucleosome-free regions
Histone chaperones regulate nucleosome dynamics
Chromatin remodeling complexes affect distribution and composition of the nucleosomes
TFs recruit histone modification enzymes (acetyl transferases, methyl transferases, kinases)
Histone variant H2A.Z promotes transcription (H2A.Z-containing nucleosomes are more labile)
Zyklusvorlesung Molekularbiologie WS 2009/10
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Wichtiges Konzept: Chromatin remodeling
Figure 7-46 Molecular Biology of the Cell (© Garland Science 2008)
Chromatin remodeling can free promoter region and thereby activate transription; it is
accomplished by chromatin remodeling complexes, histone chaperones and histone-modifying
enzymes
Zyklusvorlesung Molekularbiologie WS 2009/10
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Eukaryontischer Transkriptionszyklus
Chromatin opening
Recycling
Activators
PIC Assembly
Termination
Initiation
Eucaryotes have a larger variety of
regulatory mechanisms than bacteria
due to chromatine remodeling and
covalent modifications of histones,
TFs and Pol II
Elongation
Promoter clearance
Escape
Most important control steps:
-Chromatin opening
-Assembly of PIC
-Escape of Pol II
Zyklusvorlesung Molekularbiologie WS 2009/10
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„Offene“ und „bedeckte“ Promotoren
Most active gene promoters are not
covered by nucleosomes
Promoter region is held free by polydA:dT tracks that impare nucleosome
binding and by the H2A.Z nucleosomes
Gene expression from open promoters
is more stable
Promoters of regulated genes are typically
covered by nucleosomes
Promoter region has to be freed by
chromatin remodeling
Gene expression is stochastically variable
Zyklusvorlesung Molekularbiologie WS 2009/10
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Beispiel von einem regulierten Promotor:
PHO5-Promoter in S. cerevisiae
PHO5 gene is upregulated upon
phosphate starvation through chromatin
opening:
Activator Pho2 und histone acetyl
transferase NuA4 bind the first regulatory
sequence (UASp1) already before induction
Activator Pho4 binds at UASp1 and recruits
histone acetyl transferase SAGA
Pho2-Pho4 complex, histone acetylation by
SAGA, chromatin remodeling by Ino80 and
Swi/Snf, and histone chaperone Asf1 lead to
promoter opening and assembly of initiation
complex
Zyklusvorlesung Molekularbiologie WS 2009/10
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Mechanismen der Repression bei Eukaryonten
Figure 7-50 Molecular Biology of the Cell (© Garland Science 2008)
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Kombinatorische Integration der
Transkriptionssignale
Figure 7-58 Molecular Biology of the Cell (© Garland Science 2008)
Initiation results from integration of multiple positive and negative signals
Individual signals are integrated synergistically
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Beispiel der Signalintegration:
Segmentierung von Drosophila Embryo
What is the cause of stripe-like expression of Eve gene?
Regulators of Eve
Eve
?
Eve regulators are expressed asymmetrically, but how does this produce stripes?
Figure 7-53 Molecular Biology of the Cell (© Garland Science 2008)
Zyklusvorlesung Molekularbiologie WS 2009/10
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Beispiel der Signalintegration:
Segmentierung von Drosophila Embryo
The regulation is combinatorial and stripe-specific
Figure 7-56 Molecular Biology of the Cell (© Garland Science 2008)
Eve expression in stripe 2 is under positive regulation of Hunchback und Bicoid
and negative regulation of Giant und Krüppel
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Morphogengradienten und Genexpression
Can a tissue convert a morphogen gradient into a stripe-specific gene expression?
Particular morphogen concentration can activate only some but not the other
genes, dependent on the binding affinity:
Low concentration -> only genes with high-affinity binding sites are activated
High concentration -> all genes posessing binding sites are activated
Similar regulation can take place for temporal gradients
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Methoden der genomweiten
Transkriptionsanalyse
Classical method of expression analysis: Nothern blot
RNA (or DNA) is separated by the size on a gel,
transfered to the membrane and hybridized with
gene-specific probe
RNA -> Nothern blot
DNA -> Southern blot
Low throughput and poor quantification
Figure 8-38 Molecular Biology of the Cell (© Garland Science 2008)
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Methoden der genomweiten
Transkriptionsanalyse
Quantitative measurements of gene expression: RT-PCR
RNA
DNA
Reverse transcription
PCR
The course of PCR (amount of double-stranded DNA) is
monitored using a specific fluorescent dye
N
Differences in concentration of particular mRNA in
different samples can be calculated as 2N, with N being
the difference in the number of cycles to obtain the same
amount of product
Medium throughput, high precision
Figure 8-72 Molecular Biology of the Cell (© Garland Science 2008)
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Methoden der genomweiten
Transkriptionsanalyse
High-throughput approach: Microarrays
Figure 8-74 Molecular Biology of the Cell (© Garland Science 2008)
mRNA is converted to cDNA and labeled, and subsequently
hybridized with an array of gene-specific probes (either spotted
cDNA samples or oligonucleotides)
Differences in expression between samples are determined as
a ratio of fluorescence signals at individual spots
Up- and downregulated genes can be clustered together using
analysis software
Is not very precise, has low dynamic range
Figure 8-73 Molecular Biology of the Cell (© Garland Science 2008)
Zyklusvorlesung Molekularbiologie WS 2009/10
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Methoden der genomweiten
Transkriptionsanalyse
High-throughput approach: Next-generation (deep) sequencing
Massively parallel sequencing techniques
enable sequencing of genome-wide cellular
RNA pools
Typical sequencing reads are 30-100
nucleotides -> RNA or cDNA has to be
fragmented
A single read contains 105-107 reactions,
depending on a platform, so most RNAs are
covered by multiple „reads“ -> read occurence
for a particular gene reflects expression level
The approach is quantitative
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Methoden der genomweiten
Transkriptionsanalyse
Roche 454 sequencing
DNA fragments are coupled to beads with
specific linkers
DNA fragments are amplified on individual
beads using emulsion PCR
~400,000 individual beads are placed into well
of a microfluidic plate
Sequence reads of up to 250 bases are
produced by flowing individual
deoxynucleotides over the plate are following
PPi release through light emission
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Methoden der genomweiten
Transkriptionsanalyse
Illumina (Solexa) sequencing
DNA fragments are coupled to glass slide and
subjected to Bridge amplification
~10,000,000 individual reads of 40 bp are
produced at a time by using fluorescently labeled
removable terminator tags
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Methoden der genomweiten
Transkriptionsanalyse
Genome-wide analysis of DNA binding: Chromatin immunoprecipitation (ChIP)
Distribution of
specific TFs on a
fragment of
chromosome 1
Binding proteins are cross-linked to DNA, then DNA is sheared,
binding proteins with bound DNA fragments are purified and
amplified
Identity of bound fragments is then determined using either
Microarrays (ChIP-chip) or next-generation sequencing (ChIPSeq)
As a result, one obtains genomic distribution of transcription
factors, Pol II, nucleosomes etc