Zyklusvorlesung Molekularbiologie WS 2009/10Victor Sourjik, Seite 1 Kontrolle der Genexpression...

Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 1

Kontrolle der Genexpression

Victor Sourjik, ZMBH


Wie wird das Expressionsniveau reguliert?

Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008)


Genexpression bei Bakterien und Eukaryonten


Differences:- Genome organisation- Gene- / Transcript structure- Processing of RNA - RNA Degradation- RNA Transport- Translation

Evolutionary significance of these differences?


Genexpressionskontrolle bei Eukaryonten


Eucaryotes have more opportunities to regulate gene expression than bacteria


Vielfalt der RNA Moleküle in der Zelle

Table 6-1 Molecular Biology of the Cell (© Garland Science 2008)

What RNA polymerases transcribe what RNAs?


RNA-Polymerasen

Table 6-2 Molecular Biology of the Cell (© Garland Science 2008)

Bacteria have only one RNA polymerase

Eucaryotes have three RNA polymerases:


Transkriptionskontrolle (bei Bakterien)

Transcription

5‘ 3‘RNA

AUG UGA

DNA+1 (start site) terminator

5‘ UTR 3‘ UTRPromoter

Coding region

Initiation Elongation Termination

Initiation of transcription is the main point of regulation(both in bacteria and in eucaryotes)


Transkriptionsinitiation (bei Bakterien)

Assembly of the (pre)initiation complex (PIC) on promoter is the most frequently regulated step in bacteria and in eucaryotes

closed complex

binding

isomerisation

open complex

initiation

synthesis start

promoter clearance

elongation


„Passive“ und „aktive“ TranskriptionsregulationPassive regulation by promoter strength

Sigma 70 consensus promoter in E. coli: TTGACA...17bp...TATAAT

TTGAAA...17bp...TATAATTTCAAA...17bp...TAATAT


„Passive“ und „aktive“ TranskriptionsregulationActive regulation

Bacteria:

-Sigma Factors

-Activators

-Repressors

Eucaryotes:

-Chromatin remodeling factors-General transcription factors (GTFs)

-Activators-Repressors

-Covalent Modifications

GTFs

Pol II


Wirkungsmechanismen von Transkriptionsfaktoren (Bakterien)

binding to subunit

binding to subunit

conformational change in promoter

Activators


Wirkungsmechanismen von Transkriptionsfaktoren (Bakterien)

steric hindrance

DNA looping

modulation of

an activator

Repressors


Signalintegration (Bakterien)

repositioning

independent contacts

cooperative binding

anti-repression


Promotor- und Genstruktur bei Bakterien und Eukaryonten

+1 (start site) terminator

-35 -10UP

5‘ TTGACA...17bp...TATAAT 3‘-35 -10

Bacterial promoter

5‘ YYCAYYYYY 3‘ 5‘ AGAC 3‘

cleavage and poly-A signal

TATA box (-30)

5‘ TATAA 3‘Eucaryotic promoter

Inr (-3 to +5) DPE (+30)

GC box (-90)

CAAT box (-75)

Binding sites for activators and repressors

Proximal binding sites for activators and repressors; enhancers/UASs; silencers; insulators etc


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)


Initiationskomplex und Aktivatoren


Eucaryotic initiation complex consists of multiple general and specific TFs -> Multiple opportunities of regulation

-Mediator complex offers multiple binding sites for PIC assembly

-Eucaryotic TFs can bind to the regulatory sequences (enhancers) far upstream or downstream from the transcription start site (TSS)

-Similar to bacterial activators, many eucaryotic activators assist assembly of (pre)initiation complex

-Other activators recruit histone modifiers and chromatin remodelers


Wichtiges Konzept: DNA looping and persistence length

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!



Chromatinstruktur und Transkriptionsinitiation

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 DNADNA sequence influences nucleosome positioning„Pioneer“ TFs bind at nucleosome-free regionsHistone chaperones regulate nucleosome dynamicsChromatin remodeling complexes affect distribution and composition of the nucleosomesTFs recruit histone modification enzymes (acetyl transferases, methyl transferases, kinases)Histone variant H2A.Z promotes transcription (H2A.Z-containing nucleosomes are more labile)

GTFs

Pol II


Wichtiges Konzept: Chromatin remodeling

Chromatin remodeling can free promoter region and thereby activate transription; it is accomplished by chromatin remodeling complexes, histone chaperones and histone-modifying enzymes



Eukaryontischer Transkriptionszyklus

Chromatin opening

PIC Assembly

Initiation

Promoter clearance

Escape

Elongation

Termination

Recycling

Activators

Most important control steps: -Chromatin opening-Assembly of PIC-Escape of Pol II

Eucaryotes have a larger variety of regulatory mechanisms than bacteria due to chromatine remodeling and covalent modifications of histones, TFs and Pol II


„Offene“ und „bedeckte“ PromotorenMost active gene promoters are not covered by nucleosomes

Promoter region is held free by poly-dA: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


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


Mechanismen der Repression bei Eukaryonten



Kombinatorische Integration der Transkriptionssignale

Initiation results from integration of multiple positive and negative signals Individual signals are integrated synergistically



Beispiel der Signalintegration: Segmentierung von Drosophila Embryo


?

What is the cause of stripe-like expression of Eve gene?

Eve

Regulators of Eve

Eve regulators are expressed asymmetrically, but how does this produce stripes?


Beispiel der Signalintegration: Segmentierung von Drosophila Embryo

The regulation is combinatorial and stripe-specific


Eve expression in stripe 2 is under positive regulation of Hunchback und Bicoid and negative regulation of Giant und Krüppel


Morphogengradienten und GenexpressionCan 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 activatedHigh concentration -> all genes posessing binding sites are activated

Similar regulation can take place for temporal gradients


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 blotDNA -> Southern blot

Low throughput and poor quantification



Quantitative measurements of gene expression: RT-PCR

Reverse transcription

RNA DNA

PCR


The course of PCR (amount of double-stranded DNA) is monitored using a specific fluorescent dye

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

N



High-throughput approach: Microarrays



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



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



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

Roche 454 sequencing



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



Genome-wide analysis of DNA binding: Chromatin immunoprecipitation (ChIP)

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 (ChIP-Seq)

As a result, one obtains genomic distribution of transcription factors, Pol II, nucleosomes etc

Distribution of specific TFs on a fragment of chromosome 1

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