Transcript RNA - ZMBH
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 Victor Sourjik, Seite 5 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 7 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 Victor Sourjik, Seite 8 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 Victor Sourjik, Seite 9 „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 Victor Sourjik, Seite 10 „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 Victor Sourjik, Seite 11 Wirkungsmechanismen von Transkriptionsfaktoren (Bakterien) Activators binding to subunit binding to subunit conformation al change in promoter Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 12 Wirkungsmechanismen von Transkriptionsfaktoren (Bakterien) Repressors steric hindrance DNA looping modulation of an activator Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 13 Signalintegration (Bakterien) repositioning independent contacts cooperative binding anti-repression Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 14 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 15 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) Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 16 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) Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 17 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! Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 18 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 Victor Sourjik, Seite 19 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 Victor Sourjik, Seite 20 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 Victor Sourjik, Seite 21 „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 Victor Sourjik, Seite 22 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 Victor Sourjik, Seite 23 Mechanismen der Repression bei Eukaryonten Figure 7-50 Molecular Biology of the Cell (© Garland Science 2008) Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 24 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 25 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 Victor Sourjik, Seite 26 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 27 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 28 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) Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 29 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) Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 30 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 Victor Sourjik, Seite 31 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 32 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 33 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 Zyklusvorlesung Molekularbiologie WS 2009/10 Victor Sourjik, Seite 34 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