chapter 11: transcription in eukaryotes
DESCRIPTION
Chapter 11: Transcription in Eukaryotes. … the modern researcher in transcriptional control has much to think about. James T. Kadanoga, Cell (2004), 116:247. 11.1 Introduction. Eukaryotic gene regulation involves: DNA-protein interactions Protein-protein interactions Chromatin structure - PowerPoint PPT PresentationTRANSCRIPT
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Chapter 11:
Transcription in Eukaryotes
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… the modern researcher in transcriptional control has much to think about.
James T. Kadanoga, Cell (2004), 116:247
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11.1 Introduction
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Eukaryotic gene regulation involves:
• DNA-protein interactions
• Protein-protein interactions
• Chromatin structure
• Nuclear architecture
• Cellular compartmentalization
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11.2 Overview of transcriptional regulation
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Transcription and translation are uncoupled in eukaryotes
• Transcription takes place in the nucleus and translation takes place in the cytoplasm.
• The whole process may take hours, or in some cases, months for developmentally regulated genes.
• Gene expression can be controlled at many different levels.
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Transcription is mediated by:
• Sequence-specific DNA-binding transcription factors.
• The general RNA polymerase II (RNA pol II) transcriptional machinery.
• Coactivators and corepressors.• Elongation factors.
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Chromosomal territories and transcription factories
• Chromosome “painting” has shown that each chromosome occupies its own distinct territory in the nucleus.
• Transcription decondenses chromatin territories.
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• The DNA loops that form in decondensed regions are proposed to be associated with transcription “factories.”
• Transcriptionally active genes also appear to be preferentially associated with nuclear pore complex.
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Eukaryotes have different types of RNA polymerase
• Bacteria have one type of RNA polymerase that is responsible for transcription of all genes.
• Eukaryotes have multiple nuclear DNA-dependent RNA polymerases and organelle-specific polymerases.
• Focus here on regulation of transcription of protein-coding genes by RNA polymerase II.
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11.3 Protein-coding gene regulatory elements
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The big picture:
• Transcription factors interpret the information present in gene promoters and other regulatory elements and transmit the appropriate response to the RNA pol II transcriptional machinery.
• What turns on a particular gene in a particular cell is the unique combination of regulatory elements and the transcription factors that bind them.
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• Regulatory regions of unicellular eukaryotes such as yeast are usually only composed of short sequences located adjacent to the core promoter.
• Regulatory regions of multicellular eukaryotes are scattered over an average distance of 10 kb of genomic DNA.
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Variation in:
• Whether a particular element is present or absent.
• The number of distinct elements.
• Their orientation relative to the transcriptional start site.
• The distance between them.
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• Gene regulatory elements are specific cis-acting DNA sequences that are recognized by trans-acting transcription factors.
• Two broad categories of cis-acting regulatory elements.
– Promoter elements.
– Long-range regulatory elements.
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Structure and function of promoter elements
The gene promoter is the collection of cis-regulatory elements that are:
• Required for the initiation of transcription.
• Increase the frequency of initiation only when positioned near the transcriptional start site.
• The recognition site for RNA pol II general transcription factors.
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The gene promoter region
• Core promoter elements.
• Proximal promoter elements.
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Core promoter elements
• Approximately 60 bp DNA sequence overlapping the transcription start site.
• Serves as the recognition site for RNA pol II and the general transcription factors.
• All core promoter elements, except for BRE, are recognized by TFIID.
• A particular core promoter many contain some, all, or none of the common motifs.
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The TATA box
• First core promoter element identified in a eukaryotic protein-coding gene.
• Key experiment by Pierre Chambon and colleagues demonstrated that a viral TATA box is both necessary and sufficient for specific initiation of transcription by RNA pol II in vitro.
• Sequence database analysis suggests the TATA box is present in only 32% of potential core promoters.
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Promoter proximal elements
• Regulation of TFIID binding to the core promoter in yeast depends on an upstream activating sequence (UAS).
• Multicellular eukaryotic genes are likely to contain several promoter proximal elements.
e.g. CAAT box and the GC box
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Promoter proximal elements
• Transcription factors that bind promoter proximal elements do not always directly activate or repress transcription.
• Transcription factors may serve as “tethering elements.”
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Structure and function of long-range regulatory elements
• Additional regulatory elements in multicellular eukaryotes that can work over distances of 100 kb or more from the gene promoter.
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• Enhancers and silencers
• Insulators
• Locus control regions (LCRs)
• Matrix attachment regions (MARs)
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Enhancers and silencers
• Usually 700 to 1000 bp or more away from the start of transcription.
• Increase or repress gene promoter activity either in all tissues or in a regulated manner.
• Typically contain ~10 binding sites for several different transcription factors.
• How can you tell an enhancer from a promoter?
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Insulators
• Chromatin boundary markers.
• Enhancer or silencer blocking activity.
• Insulator elements are recognized by specific DNA-binding proteins.
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Locus control regions (LCRs)
• Organize and maintain a functional domain of active chromatin.
• Prototype LCR characterized in the mid-1980s as a cluster of DNase I-hypersensitive sites upstream of the -globin gene cluster.
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-globin gene LCR is required for high-level transcription
• Physiological levels of expression of the embryonic, fetal, and adult -globin genes only occurs when they are downstream of the LCR.
• The DNase I hypersensitive sites contain clusters of transcription factor-binding sites and interact via extensive protein-DNA and protein-protein interactions.
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Hispanic thalassemia and DNase I hypersensitive sites
• Analysis of patients with -thalassemia has led to significant advances in understanding of the LCR of the -globin gene locus.
• Partial or complete deletion of the LCR leads to reduced amounts of hemoglobin in the blood.
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Hispanic thalassemia
• ~35 kb deletion of the LCR.
• The Hispanic locus is transcriptionally silent.
• The entire region of the -globin gene cluster is DNase I-resistant.
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Analysis of DNase I sensitivity
• Transcriptionally active genes are more susceptible to deoxyribonuclease (DNase I) digestion.
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Matrix attachment regions (MARs)
• Organize the genome into loop domains.
• Typically AT rich sequences located near enhancers in 5′ and 3′ flanking sequences.
• Confer tissue specificity and developmental control of gene expression.
• “Landing platform” for transcription factors.
• Attach to the nuclear matrix.
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Position effect and long-range regulatory elements
• The function of many long-range regulatory elements was confirmed by their effect on gene expression in transgenic animals.
• Protect transgenes from the negative or positive influences exerted by chromatin at the site of integration.
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Position effect
• Expression of a transgene is unpredictable in a transgenic organism.
• Varies with the random chromosomal site of integration.
• Can long-range regulatory elements protect transgenes from position effect?
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Intron enhancers contribute to tissue-specific gene expression
• Does the enhancer located in the second intron of the apolipoprotein B gene play a role in gene regulation?
• What do the results suggest?
• Why do you think a reporter gene was used in the experiment?
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MARs promote formation of independent loop domains
• Experiment to test the importance of MARs in transcriptional regulation of the whey acidic protein (WAP) gene.
• Analyzed by Southern blot (DNA) and Northern blot (RNA).
• What do the results suggest?
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Is there a nuclear matrix?
• The nuclear matrix is operationally defined as “a branched meshwork of insoluble filamentous proteins within the nucleus that remains after digestion with high salt, nucleases, and detergent.”
• What forms the branching filaments remains unknown.
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What does the nuclear matrix do?
• Proposed to serve as a structural organizer within the cell nucleus.
• Interaction of MARs with the nucleus is proposed to organize chromatin into loop domains and maintain chromosomal territories.
• Active genes are found associated with the nuclear matrix only in cell types in which they are expressed.
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What are the components of the nuclear matrix?
• >200 types of proteins associated with the nuclear matrix.
• What forms the branching filaments remains unknown.
• General components include the heterogeneous nuclear ribonucleoprotein (hnRNP) complex proteins and the nuclear lamins.
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What are the components of the nuclear matrix?
• The nuclear lamina is a protein meshwork underlying the nuclear membrane.
• Composed of the intermediate filament proteins lamins A, B, and C.
• Internal lamins form a “veil” that branches throughout the interior of the nucleus.
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Hutchinson-Gilford progeria syndrome
• A premature aging syndrome.
• Splicing mutation in the lamin A gene.
• Patient cells have altered nuclear sizes and shapes, disrupted nuclear membranes, and extruded chromatin.
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Is there a nuclear matrix?
• Established by nuclear functions?
• Present as a structural framework which then promotes functions?
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11.4 The general transcriptional machinery
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• General, but diverse, components of large multi-protein RNA polymerase machines required for promoter recognition and the catalysis of RNA synthesis.
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Three major classes of proteins that regulate transcription
• The general (basal) transcription machinery
• Transcription factors
• Transcriptional coactivators and corepressors
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Components of the general transcription machinery
• RNA polymerase II
• General transcription factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
• Mediator
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Four major steps of transcription initiation
1.Preinitiation complex assembly
2.Initiation
3.Promoter clearance and elongation
4.Reinitiation
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Structure of RNA polymerase II
• A 12 subunit polymerase capable of synthesizing RNA and proofreading nascent transcript.
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Crystal structure for Saccharomyces cerevisiae RNA polymerase II
• 12 subunits total (Rpb1 to 12).
• 10 subunit catalytic core.
• Heterodimer of Rpb4 and Rpb7.
• Unstructured C-terminal domain (CTD) of Rpb1 is not seen by X-ray crystallography.
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RNA polymerase II catalytic core
• The wall prevents straight passage of nucleic acids through the cleft.
• The RNA-DNA hybrid is nearly 90 to that of the entering DNA duplex.
• A pore beneath the active site widens towards the outside like a funnel and includes two Mg2+
binding sites.
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• Positively charge “cleft” occupied by nucleic acids.
• One side of cleft is formed by a massive, mobile “clamp.”
• The active site is formed between the clamp, a “bridge helix” and a “wall”.
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RNA polymerase II C-terminal domain (CTD)
• Tail-like feature of the largest subunit.
• Consists of up to 52 heptapeptide repeats.
• Undergoes dynamic phosphorylation of serine residues at positions 2 and 5 in the repeats.
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General transcription factors and preinitiation complex formation
• A set of five general transcription factors, denoted TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
• Responsible for promoter recognition and unwinding of promoter DNA.
• Nomenclature denotes “transcription factor for RNA polymerase II.”
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• RNA polymerase II is absolutely dependent on these auxiliary transcription factors for the initiation of transcription.
• TFIIA and its subunit TFIIJ are not absolutely required for transcription initiation in vitro, so are not considered general transcription factors.
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• Transcription initiation requires an unphosphorylated CTD.
• Assembling the general transcription apparatus involves a series of highly ordered steps.
• Binding of TFIID provides a platform to recruit other general transcription factors and RNA pol II to the promoter.
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TFIID recruits the rest of the transcriptional machinery
• Binding of TFIID to the core promoter is a critical rate limiting step.
• TFIID is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs)
• TBP contains an antiparallel -sheet that sits on the DNA like a saddle in the minor groove and bends the DNA.
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TFIIB orients the complex on the promoter
• TFIIB binds to one end of TBP and to a GC-rich DNA sequence after the TATA motif.
• The TFIID-TBP-DNA complex “signposts” the direction for the start of transcription.
• The complex indicates which strand acts as the template.
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TFIIE, TFIIF, and TFIIH binding completes the preinitiation complex
formation
• RNA polymerase II joins the assemblage in association with TFIIF and Mediator.
• TFIIE binds and recruits TFIIH.
• Promoter melting is mediated by the helicase activity of TFIIH.
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• Unwinding is followed by “capture” of the nontemplate strand by TFIIF.
• The template strand descends into the active site of RNA polymerase II.
• Because of the conserved spacing from the TATA box to the transcription start site, the start site is positioned in the polymerase active site.
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TFIIH has both cyclin-dependent kinase activity and helicase activity
• Transcription elongation requires a phosphorylated CTD.
• TFIIH is the kinase that phosphorylates the CTD of RNA pol II.
• Landmark experiments showed that purified TFIIH could convert the unphosphorylated polymerase to its phosphorylated form in vitro.
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• The ATP-dependent helicase activity of TFIIH was demonstrated using an in vitro assay.
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Mediator: a molecular bridge
• A 20-subunit complex which transduces regulatory information from the activator and repressor proteins to RNA pol II.
• Mediator serves as a molecular bridge between the transactivation domain of various transcription factors and RNA pol II.
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The discovery of Mediator
• In vitro transcription assays could support basal transcription but were not responsive to transcriptional activators.
– Minimum set of general transcription factors– Purified core RNA pol II– G-less cassette assay
• Mediator was shown to be required for activator-responsive transcription.
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• Mediator is expressed ubiquitously in eukaryotes.
• At least seven different mammalian Mediator complexes with 25-30 protein subunits.
• Comprised of head, middle, and tail modules according to electron microscopy.
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• Conserved flexible hinge in the middle of the MED7/MED 21 heterodimer may promote changes in Mediator structure upon binding RNA pol II or transcription factors.
• How does Mediator reach regulatory elements that are so far away from the gene promoter?
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Initiation of transcription
• Assembly of the preinitiation complex.
• A period of abortive initiation.
• Promoter clearance.
• Elongation.
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Abortive initiation
• RNA polymerase II synthesizes a series of short transcripts
• As it moves, the polymerase holds the DNA strands apart forming a transcription bubble.
• A transcript of >10 nucleotides and bubble collapse lead to promoter clearance.
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Promoter clearance
• Requires phosphorylation of the C-terminal domain (CTD) of RNA pol II.
• Phosphorylation helps RNA pol II to leave behind most of the general transcription factors.
• TFIID remains bound at the promoter and allows the rapid formation of a new preinitiation complex.
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Once phosphorylated, RNA polymerase II can:
• Unwind DNA.
• Polymerize RNA.
• Proofread.
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11.5 The role of specific transcription factors in gene
regulation
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Transcription factors influence that rate of transcription of specific genes either positively or negatively by:
• Specific interactions with DNA regulatory elements.
• Interaction with other proteins.
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• Gene regulation at the transcriptional level generally occurs via changes in the amounts or activities of transcription factors.
• The genes encoding transcription factors may be activated or repressed by other regulatory proteins.
• Transcription factors themselves may be activated or deactivated by proteolysis, covalent modifications, ligand binding, etc.
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Transcription factors mediate gene-specific transcriptional activation
or repression
• Transcription factors that serve as repressors block the general transcription machinery.
• Transcription factors that serve as activators increase the rate of transcription by several mechanism.
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Mechanisms of action of transcriptional activators
1.Stimulate the recruitment and binding of general transcription factors and RNA polymerase II.
2. Induce a conformational change or post-translational modification that stimulates the enzymatic activity of the general transcription machinery.
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3. Interact with chromatin remodeling and modification complexes to increase DNA accessibility to the transcription machinery.
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Transcription factors are modular proteins
• Composed of separable, functional domains.
• The three major domains are a DNA-binding domain, a transactivation domain, and a dimerization domain.
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DNA-binding domain motifs
• Hundreds of protein-DNA complexes have been analyzed by X-ray crystallography.
• NMR spectroscopy has been used to study complexes in solution.
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• High affinity binding is dependent on overall 3-D shape and formation of specific hydrogen bonds.
• Loss of just a few hydrogen bonds or hydrophobic contacts from a protein-DNA complex will usually result in a large loss of specificity.
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• The most common recognition pattern is an interaction between an -helical domain of the protein and about 5 bp within the major groove of the DNA double helix.
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Some of the most common DNA-binding domain motifs:
• Helix-turn-helix
• Zinc finger
• Basic leucine zipper
• Basic helix-loop-helix
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Helix-turn-helix (HTH)
• The first DNA-binding domain to be well characterized.
• The classic HTH is composed of three core -helices.
• The third “recognition” helix inserts into the major groove of the DNA.
• Other variant forms may contain additional features, such as in the winged HTH motif
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Homeoboxes and homeodomains
• In the late 1970s, developmental biologists were studying the genes regulating pattern formation in the developing Drosophila embyro.
• Discovery of homeobox genes in Drosophila.
• Later discovered in many other organisms from humans to the plant Arabidopsis.
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• Homeobox genes encode transcription factors with a homeodomain that regulate many developmental programs.
• The homeodomain is a variant of the classic HTH.
• The best known homeobox gene subclass is the Hox family.
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Homeotic mutations
• Mutations in some of these homeobox genes result in homeotic transformation.
• A homeotic mutation is a mutation that transforms one body part into another part:
– Antennapaedia homeotic mutant in which the antennae are transformed into legs.
– In humans, a mutation in the Hoxd13 gene results in duplication of a digit and development of six fingers.
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The Hox genes of Drosophila
• Eight Hox genes regulate the identity of regions within the adult fruitfly and embryo.
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Colinear expression of homeobox genes
• Homeobox genes are organized in clusters.
• Differential gene expression in which an expression gradient is achieved either spatially, temporally, or quantitatively depending on the location of each gene in the cluster.
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Spatial colinearity
• The position of a gene in a cluster correlates with its expression domain.
• 5′ and 3′ Hox genes are typically expressed in the posterior and anterior portions of developing embryos, respectively.
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Temporal colinearity
• Genes at one end of the cluster are turned on first and genes at the opposite end are turned on last.
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Quantitative colinearity
• The first gene in a cluster displays a maximum level of expression and downstream genes exhibit progressively lower expression.
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The Rhox genes of mouse
• A cluster of twelve homeobox genes on the X-chromosome.
• Important regulatory role in reproduction.
• The genes are expressed in the order they occur on the chromosome during sperm differentiation.
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Polycomb group proteins silence homeobox genes
• Mediate gene silencing by altering the higher order structure of chromatin.
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Zinc finger (Zif)
• One of the most prevalent DNA-binding motifs.
• First described in 1985 for Xenopus laevis TFIIIA.
• A “finger” is formed by interspersed cysteines and/or histidines that covalently bind a central zinc (Zn2+) ion.
• The finger inserts its -helical portion into the major groove of the DNA.
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• The number of fingers is variable between different transcription factors.
• Classic finger: Cys2-His2 pattern.
• Nuclear receptors have two fingers of a Cys2-Cys2 pattern.
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Zinc finger DNA-binding domain of the glucocorticoid receptor (GR)
• Three to four amino acids at the base of the first finger confer specificity of DNA binding.
• A dimerization domain near the base of the second finger is the region that interacts with another GR to form a homodimer.
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• Each protein in the pair recognizes half of a two-part DNA regulatory element called a glucocorticoid response element (GRE).
• GR distinguishes both the base sequence and the spacing of the half sites.
• Other nuclear receptors share the same sequence, but have different spacing in between.
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Greig cephalopolysyndactyly syndrome and Sonic hedgehog signaling
• Autosomal dominant disease.
• Physical abnormalities affecting the fingers, toes, head, and face.
– Polydactyly: extra fingers or toes.– Syndactyly: webbing and/or fusion of the
fingers and toes.
• Mutations in the GLI3 gene.
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• Mutation in the zinc finger protein GLI3 disrupts the Sonic hedgehog signaling pathway during development.
• Sonic hedgehog is one of three vertebrate homologs to the Drosophila hedgehog gene
• Named for “Sonic the Hedgehog,” a character in a popular video game.
• Encodes a secreted protein.
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Sonic hedgehog signaling
• The transmembrane receptor protein, Patched-1 (PTC-1) inhibits downstream signaling by interaction with Smoothened.
• When the Sonic hedgehog signal (SHH) binds PTC-1 it relieves repression of Smoothened.
• Leads to the activation and repression of target genes by GLI family transcription factors.
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Defective histone acetyltransferases in Rubinstein-Taybi syndrome
• Rubinstein-Taybi syndrome is a rare autosomal dominant disease characterized by facial abnormalities, broad digits, stunted growth, and mental retardation.
• The disease is due to mutations in the gene coding for CBP, a coactivator with histone acetyltransferase activity.
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Basic leucine zipper (bZIP)
• bZIP motif is a stretch of amino acids that folds into a long -helix with leucines in every seventh position, forming a hydrophobic “stripe.”
• bZIP motif is not the DNA binding domain.
• Plays an indirect structural role in DNA binding by facilitating dimerization.
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• Two polypeptides with a bZIP motif form a “coiled-coil” Y-shaped structure.
– Homodimer: two of the same polypeptide.
– Heterodimer: two different polypeptides.
• One end of each -helix protrudes into the major groove of the DNA.
• The two basic binding regions contact the DNA.
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• The transcription factor AP-1 is a heterodimer of Fos and Jun.
• The bZIP domains are essential for binding.
• Jun can form both homodimers and heterodimers.
• Fos can only form heterodimers.
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Basic helix-loop-helix (bHLH)
• Forms two amphipathic helices, containing all the charged amino acids on one side of the helix.
• Helices are separated by a loop.
• bHLH motif is not the DNA binding domain.
• Plays an indirect structural role in DNA binding by facilitating dimerization.
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• When the BHLH protein Max binds to Myc, the Myc-Max complex is a transcriptional activator.
• When Max binds to Mad, the Mad-Max complex is a transcriptional repressor.
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Transactivation domain
• Activates transcription via protein-protein interactions.
• Structurally more elusive than DNA-binding motifs.
– “Acid blobs”– Glutamine-rich regions– Proline-rich regions– Hydrophobic -sheets.
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Dimerization domain
• The majority of transcription factors bind DNA as homodimers or heterodimers.
• What are two well-characterized dimerization domains?
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11.6 Transcriptional coactivators and corepressors
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Increase or decrease transcriptional activity without binding DNA directly by:
• Serving as scaffolds for recruitment of proteins with enzymatic activity.
• Having enzymatic activity themselves for altering chromatin activity.
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• More difficult to study compared with transcription factors.
• In general, assays for protein-protein interactions are more difficult to perform than techniques for studying DNA-protein interactions.
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Two main classes of coactivators
• Chromatin modification complexes.
• Chromatin remodeling complexes.
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Chromatin modification complexes
• Multiprotein complexes that modify histones post-translationally, in ways that allow greater access of other proteins to DNA.
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Post-translational modification of histone N-terminal tails
• The N-terminal tails of histones H2A, H2B, H3, and H4 are subject to a wide range of post-translational modifications.
• Function as master on/off switches that determine whether a gene is active or inactive.
• Recognition landmarks by other proteins that bind chromatin.
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Four major types of modification
• Acetylation of lysines• Methylation of lysines and arginines• Ubiquitinylation of lysines• Phosphorylation of serines and threonines
Two less common types
• ADP-ribosylation of glutamic acid• Sumoylation of lysines
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• Levels of specific histone modifications or “marks” are maintained by balanced activities of modifying and demodifying enzymes.
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Histone acetyltransferases
• Histone acetyltransferase (HAT) directs acetylation of histones at lysine residues.
• Histone deacetylase (HDAC) catalyzes removal of acetyl groups.
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• The addition of the negatively charged acetyl group reduces the overall positive charge of the histones.
• Decreased affinity of the histone tails for the negatively charged DNA.
• Acetylation of lysines provides a specific binding surface that can either recruit repressors or activators of gene activity.
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Histone methyltransferases
• Histone methyltransferase (HMT) directs methylation of histones on both lysine and arginine residues.
• Histone demethylase removes methyl groups.
e.g. lysine-specific demethylase 1 (LSD-1)
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• The methyl groups increase the bulk of histone tails but do not alter the electric charge.
• Histone methylation is linked to both activation and repression of transcription.
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Ubiquitin-conjugating enzymes
• A ubiquitin-conjugating enzyme adds one ubiquitin to a lysine residue.
• Isopeptidase removes ubiquitin.
• Monoubiquitinylation of H2B is associated with activation or silencing.
• Monoubiquitinylation of linker histone H1 leads to its release from DNA and gene activation.
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Kinases
• A specific kinase adds a phosphate group to one or more serine or threonine amino acids, adding a negative charge.
• Phosphatase removes phosphate groups.
• Phosphorylation of histone H3 or the linker histone H1 is associated with the activation of specific genes.
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ADP-ribosyltransferases
• ADP-ribosyltransferase adds an ADP-ribose from NAD+ to a glutamic acid residue.
• ADP-ribosylhydrolase removes ADP-ribose.
• MacroH2A a histone variant associated with X-chromosome inactivation may function in ADP-ribosylation of histones.
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SUMO-conjugating enzymes
• Small ubiquitin-like modifier (SUMO) is a 97 amino acid protein that has 20% identity with ubiquitin.
• SUMO-conjugating enzymes add SUMO to lysines.
• SUMO-specific proteases remove SUMO.
• In most cases, sumoylation is associated with transcription repression.
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Linker histone variants
• Mammals contain eight histone H1 subtypes
• H1a through H1e and H1 in somatic cells
• Two germ-cell specific subtypes, H1t and H1oo.
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Is there a histone code?
• Histone modifications are used as recognition landmarks by other proteins.
• Chromodomain motif targets proteins to methylated lysines
• Bromodomain motif targets proteins to acetlyated lysines.
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The histone code hypothesis
• Covalent post-translational modifications of histone tails are read by the cell and lead to a complex, combinatorial transcriptional output.
• The hypothesis continues to be a subject of much debate:
– Some researchers conclude that if there is a “code” it is a simple one and not combinatorial.
– Other researchers conclude that histone modifications are more like a “complex language.”
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Depression of the MyoD gene by the linker histone H1b
• The Msx1 homeodomain protein forms a complex with H1b.
• The complex binds to an enhancer element in the MyoD gene and inhibits gene expression.
• This prevents differentiation of muscle progenitor cells.
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Chromatin remodeling complexes
• Use the energy from ATP hydrolysis to change the contacts between histones and DNA.
• Allow transcription factors to bind to DNA regulatory elements.
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Mediate at least four different changes in chromatin structure:
• Nucleosome sliding
• Remodeled nucleosomes
• Nucleosome displacement
• Nucleosome replacement
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Three main families defined by a unique subunit composition and the presence of a distinct ATPase
• SWI/SNF complex family
• ISWI complex family
• SWR1 complex family
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Mode of action of SWI/SNF: nucleosome sliding and disassembly
• The SWI/SNF complex from the budding yeast was the first chromatin remodeling complex to be characterized.
• 2 MDa complex composed of at least 11 different polypeptides.
• Many other chromatin remodeling factors in this family have been identified, from Drosophila to humans.
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ISWI chromatin remodeling complexes slide histone octamers
along DNA
• Change the position of a nucleosome on the DNA.
• Relocate nucleosomes by sliding the histone octamers along the DNA without perturbation of their structure.
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Histone replacement with a variant histone by the SWR1 chromatin
remodeling complex
• Histone replacement with a variant histone in the core octamer.
• Can replace histone H2A-H2B dimers with H2A.Z-H2B dimers.
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11.7 Transcription complex assembly: the enhanceosome model versus the
“hit-and-run” model
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• The dynamic process by which transcription factors and coactivators interact on DNA to activate transcription is the subject of much study.
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Order of recruitment of various proteins that regulate transcription
• No general rule for the order of recruitment
• Gene-specific order of events.
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Yeast HO promoter:
• The Swi5p transcription factor recruits SWI/SNF and a HAT complex, followed by a second transcription factor, SBF, before assembly of the preinitiation complex.
Human -antitrypsin gene promoter:
• Multiple HAT complexes and SWI/SNF are recruited after preinitiation complex assembly.
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Two models for binding of transcription factors and assembly of transcription complexes
• Enhanceosome model
• Hit and run model
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Enhanceosome model
• Interactions among transcription factors promote their cooperative stepwise assembly on DNA.
• Exceptionally stable complex.
• Example: interferon- promoter.
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Hit and run model
• The “hit”: Transcriptional activation reflects the probability that all components required for activation will meet at a certain site.
• The “run”: Binding is transient.
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• Transient and dynamic binding can be observed by fluorescence recovery after photobleaching (FRAP) experiments.
• Example: The glucocortiocoid receptor binds and unbinds to chromatin in cycles of only a few seconds.
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Merging of models
• The principles of combinatorial interaction and complex stability apply to hit and run models even if the complex itself has a very limited lifetime.
• Example: Interaction between high mobility group box 1 protein (HMGB1) and the glucocorticoid receptor lengthens each other’s residence time on chromatin.
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11.8 Transcription elongation through nucleosomes
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• Pausing of RNA pol II in early elongation plays an important role in gene regulation.
• In Drosophila, RNA pol II synthesizes 25-50 nt of RNA prior to heat shock and then pauses.
• Heat-shock jump starts the polymerase and it immediately begins elongation.
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Transcription elongation
RNA transcript synthesis by RNA pol II occurs by a four step cycle:
1.A nucleoside triphosphate (NTP) enters the entry (E) site beneath the active center.
2.The NTP rotates into the nucleotide addition (A) site and is checked for mismatches.
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3. Pretranslocation: phosphodiester bond formation.
4. Translocation and post-translocation: the NTP just added to the RNA transcript moves into the next position, leaving the A site open for entry of another NTP.
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Proofreading and backtracking
• RNA polymerase has one “tunable active site” that switches between RNA synthesis and cleavage.
• How does this compare with DNA polymerase proofreading?
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Proofreading
• RNA polymerization and cleavage both require metal ion “A” (e.g. Mg2+ ) in the active site.
• The differential positioning of metal ion “B” switches activity from polymerization to cleavage.
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Backtracking
• When transcribing, if RNA pol II encounters an arrest site, the polymerase pauses.
• The polymerase then backtracks, and with the help of TFIIS cleaves the unpaired 3′ end of the transcript.
• Transcription then continues on past the arrest site.
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Role of TFIIS in RNA cleavage
• TFIIS is proposed to insert an acidic -hairpin loop into the active center of RNA pol II to position metal B and a nucleophilic water molecule for RNA cleavage.
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Transcription elongation through the nucleosomal barrier
• Most of the factors discussed so far are required for the initiation of transcription but not for elongation.
• RNA polymerase encounters a nucleosome approximately every 200 bp.
• Other factors are needed for the polymerase to move through the nucleosomal array.
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Two distinct mechanisms for the progression of RNA polymerases through chromatin
• Nucleosome mobilization or “octamer transfer.”
• Histone H2A-H2B dimer removal.
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Nucleosome mobilization
• Mechanism for RNA polymerase III and bacteriophage SP6 RNA polymerase.
• Nucleosomes are translocated without release of the core octamer.
• May be facilitated by the elongation factor FACT.
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Histone H2A-H2B dimer removal
• Mechanism for RNA polymerase II.
• Requires a number of auxiliary factors, including:
– FACT (facilitates chromatin transcription)– Elongator– TFIIS
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FACT promotes nucleosome displacement
• Experiments have shown that FACT mediates displacement of an H2A-H2B dimer, leaving a “hexasome” on the DNA.
• FACT helps to redeposit the dimer after passage of RNA pol II.
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Elongator facilitates transcript elongation
• Human Elongator is composed of six subunits, including a HAT with specificity for histone H3.
• Interacts directly with RNA pol II and facilitates transcription.
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TFIIS relieves transcriptional arrest
• The elongation factor, TFIIS, facilitates passage of RNA pol II through regions of DNA that can cause transcription arrest.
– AT-rich sequences.
– The presence of DNA-binding proteins.
– Lesions in the transcribed strand.
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Defects in Elongator and familial dysautonomia
• Familial dysautonomia is a disorder of the sensory and autonomic nervous system.
• Autosomal recessive.
• Common in Ashkenazi Jewish populations.
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Symptoms of familial dysautonomia
• Absence of tears when crying.
• Decreased perception of heat, pain, taste:– e.g. an individual leaning on a pot of boiling
water may not feel it and could be seriously burned.
• Breath-holding episodes.
• Vomiting in response to stress.
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A defect in the IKBKAP gene causes familial dysautonomia
• Mutations in IKBKAP, the gene encoding one subunit of Elongator.
• Splice site mutation results in tissue-specific exon-skipping.
• Brain cells are particularly sensitive.
• Link between gene mutation and symptoms is not yet clear.
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11.9 Nuclear import and export of proteins
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• Protein synthesis occurs in the cytoplasm.
• How do transcription factors get into the nucleus?
• Trafficking between the nucleus and cytoplasm occurs via the nuclear pore complexes (NPCs).
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The nuclear pore complex
• Large multiprotein complexes embedded in the nuclear envelope.
• Eight-fold radial symmetry.
• Composed of about 30 different nucleoporins.
• Central 9-11 nm channel that can increase to an effective diameter of 45-50 nm.
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• The NPC allows bidirectional passive diffusion of ions and small molecules.
• Nuclear proteins, RNAs, and RNPs larger than ~9 nm in diameter (~40-60 kD) selectively, and actively, enter and exit the nucleus by a signal-mediated and energy-dependent process.
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• Proteins are targeted to the nucleus by a specific amino acid sequence called a nuclear localization sequence (NLS).
• Some shuttling proteins also have a nuclear export sequence (NES).
• Nuclear import and export are mediated by a family of soluble receptors, collectively called karyopherins.
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• The presence of several different NLSs and NESs and multiple karyopherins suggest the existence of multiple pathways for nuclear localization.
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Karyopherins mediate nuclear import and export
• Karyopherins are composed of helical molecular motifs called HEAT repeats.
• Form highly flexible superhelical or “snail-like” structures.
• Karyopherins that mediate nuclear import are called importins.
• Karyopherins that mediate export are called exportins.
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• Importin-1 is one of the predominant karyopherins that drives import.
• Most cargoes require the adaptor protein importin
• Seven different importin- adapters have been characterized in mammals.
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Nuclear import pathway
• Signal sequences targeting proteins to the endoplasmic reticulum or mitochondrion are removed during transit.
• In contrast, nuclear proteins retain their NLSs.
• There is no real consensus sequence, but many are lysine and arginine-rich.
• Some are bipartite.
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The process of nuclear import involves three main steps:
1.Cargo recognition and docking.
2.Translocation through the nuclear pore complex.
3.Cargo release and receptor recycling.
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Cargo recognition and docking
• The import receptor for the classic, lysine/arginine-rich NLS is a complex of importin- and importin-1.
• Importin binds directly to the NLS of the cargo.
• Importin 1 binds to both importin and to NPC proteins.
• This step does not require energy.
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Translocation through the nuclear pore complex
• The exact mechanism for cargo translocation is poorly understand.
• Weak hydrophobic interactions between importins and the FG repeat domains of nucleoporins seem to be essential.
• This step does not require energy and transport occurs via diffusion.
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Two current models for translocation through the nuclear pore complex
• Affinity gate model
• Selective phase model
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• Nuclear import (and export) occurs against a concentration gradient .
• The energy source and directional cue are provided by the small GTPase Ran.
• RanGTP is at a high concentration within the nucleus and a low concentration within the cytoplasm.
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• Ran belongs to a superfamily of GTP-binding proteins that act as molecular switches cycling between GDP- and GTP-bound states.
• The conversion from the GDP- to GTP-bound state involves nucleotide exchange.
• The conversion from the GTP- to GDP-bound state occurs by removal of the terminal phosphate from the bound GTP.
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• The RanGTP gradient is maintained by an asymmetric distribution of auxiliary factors.
• The Ran guanosine-nucleotide exchange factor RanGEF is a resident nuclear protein that binds chromatin.
• The Ran-specific GTPase-activating protein (RanGAP) is excluded from the nucleus and is at its highest concentration at the outer face of the NPC.
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Cargo release and receptor recycling
• Once the cargo-import receptor complex reaches the nuclear side of the NPC, RanGTP binds to the importin and dislodges it from the cargo.
• The RanGTP-importin complex is exported to the cytoplasm.
• After GTP hydrolysis, the export complex dissociates.
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• The importins are recycled for another round of import.
• RanGDP is rapidly imported into the nucleus by transport factor NTF2.
• RanGDP is converted to RanGTP by nucleotide exchange with the aid of RanGEF.
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Nuclear export pathway
• The best characterized nuclear export sequences (NESs) are leucine-rich.
• First described in the HIV-1 Rev protein.
• The classic Rev-type NES functions by interaction with the export factor CRM1.
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• Exportins bind to their cargo in the nucleus in the presence of RanGTP.
• In the cytoplasm, the “spring-loaded” complex disassembles spontaneously upon GTP hydrolysis.
• The export receptor is recycled.
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11.10 Regulated nuclear import and signal transduction
pathways
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• Localization of a protein at “steady” state depends on the balance between import, retention, and export, and which signal is dominant.
• A transcription factor may be sequestered in the cytoplasm until an extracellular signal induces its nuclear import.
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Regulated nuclear import of NF-B
• NF- B is a dimeric transcription factor that is a central mediator of the human stess response.
• Plays a key role in regulating cell division, apoptosis, and immune and inflammatory responses.
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The events leading to signal-mediated nuclear import of NF-B involve three main stages:
1.Cytoplasmic retention of NF-B by I-B.
2.A signal transduction pathway that induces phosphorylation and degradation of I-B.
3. I-B degradation results in exposure of the NLS on NF-B, allowing nuclear import of NF-B.
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Cytoplasmic retention by I-B
• In a resting B lymphocyte, NF-B subunits (e.g. p50 and p65) form homodimers or heterodimers in the cytoplasm.
• The dimers are retained in the cytoplasm by an anchor protein called I-B.
• I-B contains a stretch of 5-7 ankyrin repeat domains that mask the NLS of NF-B.
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Signal transduction pathways induce phosphorylation and degradation
of I-B
• Upon receipt of an extracellular signal (e.g. tumor necrosis factor ), a signal transduction pathway is triggered.
• Leads to activation of the serine-specific I-B kinase (IKK) complex.
• I-B is phosphorylated at two conserved serines.
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I-B degradation results in exposure of the NLS on NF-B
• Phosphorylation of I-B triggers release from NF-B and proteasome-mediated degradation.
• The NLS of NF-B is exposed.
• In the nucleus NF-B activates target genes by binding to specific DNA regulatory elements.
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Regulated nuclear import of the glucocorticoid receptor
• The glucocorticoid receptor mediates a highly abbreviated signal transduction pathway.
• The receptor for the extracellular signal is cytoplasmic and carries the signal directly to the nucleus.
• Leads to many diverse cellular responses ranging from increases in blood sugar to anti-inflammatory actions.
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In the absence of hormone
• GR remains cytoplasmic, bound in a complex with Hsp90 and p59.
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In the presence of hormone
• Ligand-induced conformational change releases GR from the Hsp90-p59 complex.
• Recent evidence suggests that Hsp90 may play a role in efficient nuclear entry.
• Two GRs form a homodimer, which undergoes nuclear import.
• GR activates target genes by binding to specific DNA regulatory elements.