Molecular Biology of the Cell

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1 Alberts Johnson Lewis Raff Roberts Walter Molecular Biology of the Cell Fifth Edition Chapter 7 Control of Gene Expression Copyright Garland Science 2008 A neuron and a lymphocyte share the same genome Figure 7-1 Molecular Biology of the Cell ( Garland Science 2008)

2 If DNA were altered irreversibly during development, the chromosomes of a differentiated cell would be incapable of guiding the development of the whole organism. Biologists originally suspected that genes might be selectively lost when a cell becomes specialized. The different cell types of a multicellular organism contain the same DNA All the cells contain the same genome. Cell differentiation is achieved by changes in gene expression. Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism Figure 7-2a Molecular Biology of the Cell ( Garland Science 2008) Figure 7-2b Molecular Biology of the Cell ( Garland Science 2008)

3 Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism. What makes cells express genes quite different from each other? Figure 7-2c Molecular Biology of the Cell ( Garland Science 2008) Berg Tymoczko Stryer Different cell types produce different sets of proteins A typical differentiated human cell expresses perhaps 5,000-15,000 genes from a repertoire of about 25,000. Biochemistry Seventh Edition CHAPTER 32 The Control of Gene Expression in Eukaryotes Copyright 2012 by W. H. Freeman and Company

4 Differences in mrna expression patterns among different types of human cancer cells Figure 7-3 Molecular Biology of the Cell ( Garland Science 2008) (red bar indicates that the given gene is transcribed at high level) Differences in the proteins expressed by two human tissues A cell can change the expression of its genes in response to external signals Extracellular cues (starvation or intense exercise) Glucocorticoid hormones (liver cell) Increase the production of glucose from amino acids and other small molecules. Induce enzymes such as tyrosine aminotransferase (which helps to convert tyrosine to glucose). Glucocorticoid hormones (fat cells) Reduce production of tyrosine aminotransferse. Some other cells do not respond to glucocorticoids at all. Figure 7-4 Molecular Biology of the Cell ( Garland Science 2008)

5 Endocrine system adapted fromhttp://catalog.flatworldknowledge.com/bookhub/reader/22209?e=stangor-ch03_s04

6 Protein kinase networks integrate information to control complex cell behaviors Therefore, gene expression is affected and regulated by a variety of external signals Gene expression can be regulated at many of the steps in the pathway from DNA to RNA to protein REVIEW Polymerase II also requires activator, mediator, and chromatin modifying proteins Figure 7-5 Molecular Biology of the Cell ( Garland Science 2008)

7 How Transcriptional Switches Work What makes cells express genes quite different from each other? Transcription is controlled by proteins binding to regulatory DNA sequences Regulatory DNA sequences In addition to the promoter, nearly all genes have the regulatory DNA sequences. Used to switch the gene on or off. Short (10 nt) : Simple gene switches. Very long (10 kbp) : molecular microprocessors. Recognized by gene regulatory proteins. Combination of a DNA sequence and its associated regulatory protein molecules Acts as the switch to control transcription. Table 7-1 Molecular Biology of the Cell ( Garland Science 2008)

8 Gene regulatory protein (Transcription Factor) Several thousand proteins in human. Bind to the major groove of a DNA helix. Contain a variety of DNA-binding motifs. 1. Homeodomain : 3 linked α helices. 2. Zinc finger motif : an α helix and a β sheet. 3. Leucine zipper motif : two α helices. Figure 7-6 Molecular Biology of the Cell ( Garland Science 2008) A Gene Regulatory Protein Binds to the major groove of a DNA helix. Gene Regulatory Proteins Contain DNA-binding Motifs Homeodomain Homeodomain Zinc finger motif Leucine zipper motif Figure 7-9 Molecular Biology of the Cell ( Garland Science 2008)

9 Homeodomain A structure motif in many eucaryotic DNA-binding proteins. Three linked α helices. Most of the contacts are made by helix 3. Helix 3 makes important contact with the major groove of DNA (ex. The Asn contacts an adenine). A member of the helix-turn-helix family. Figure 7-13 Molecular Biology of the Cell ( Garland Science 2008) Homeodomain Homeodomain Figure 7-11 Molecular Biology of the Cell ( Garland Science 2008)

10 One type of zinc finger protein (Cys-Cys-His-His family) Zinc Finger Domain Built from an α helix and a β sheet by a molecule of zinc. Often found in clusters. α helix contacts the DNA bases. Recognize DNA using three zinc fingers of the Cys-Cys-His- His type. Figure 7-14 Molecular Biology of the Cell ( Garland Science 2008) DNA binding by a zinc finger protein A dimer of the zinc finger domain of the intracellular receptor family Figure 7-15 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-16 Molecular Biology of the Cell ( Garland Science 2008)

11 Zinc Finger Domain p53 uses loops rather than α helices and β sheets (The folding of p53 requires a zinc, but the way in which the zinc is grasped is completely different from that of the zinc finger proteins) Figure 7-18 Molecular Biology of the Cell ( Garland Science 2008) Leucine Zipper Domain Two α helices. Bind to DNA as dimers. Each motif makes many contacts with DNA. Figure 7-19 Molecular Biology of the Cell ( Garland Science 2008)

12 Leucine Zipper Domain 1. A gel-mobility shift assay (This readily detects sequence-specific DNAbinding proteins) An electrophoretic mobility shift assay (EMSA) A mobility shift electrophoresis A gel shift assay A band shift assay A gel retardation assay Figure 7-27a Molecular Biology of the Cell ( Garland Science 2008) Figure 7-27b Molecular Biology of the Cell ( Garland Science 2008) Figure 7-30 Molecular Biology of the Cell ( Garland Science 2008)

13 2. DNA affinity chromatography 3. DNA footprinting Figure 7-28 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-29a Molecular Biology of the Cell ( Garland Science 2008) Phylogenetic footprinting Figure 7-29b Molecular Biology of the Cell ( Garland Science 2008) Figure 7-31 Molecular Biology of the Cell ( Garland Science 2008)

14 4. Chromatin Immunoprecipitation (ChIP) (This allows the identification of all the sites in a genome that a gene regulatory protein occupies in vivo) A gene regulatory circuit (S. cerevisiae) ChIP ChIP-chip (ChIP microarray) ChIP-Seq Figure 7-32 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-33 Molecular Biology of the Cell ( Garland Science 2008) Transcriptional switches allow cells to respond to changes in the environment (Procaryotic cell) REVIEW Bacterium E. coli : single circular DNA of 4.6 x 106 nt. Regulate the expression : food sources. Example: Tryptophan Operon Operon: A set of genes a single mrna. Common in bacteria. 5 genes code for enzymes for tryptophan. Figure 7-34 Molecular Biology of the Cell ( Garland Science 2008)

15 Tryptophan Repressor Repressor inactive. RNA polymerase transcribes Try genes. Repressor active. Binds to the operator. Blocks the binding of RNA polymerase. Figure 7-35 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-36 Molecular Biology of the Cell ( Garland Science 2008) Homeodomain *Constitutive gene (cf. inducible gene): Continuously transcribed at a low level without regulation of gene expression (ex. Trp repressor). The bacterium can respond very rapidly to the rise in tryptophan concentration. Allosteric protein: Binding of Trp causes a subtle change in its three-dimensional structure of repressors. Repressors can now bind to the operator DNA. Figure 7-11 Molecular Biology of the Cell ( Garland Science 2008)

16 Repressors turn genes off, activators turn them on Activator Activator Switch / on. Regulatory sequence. Initiate transcription. Controlled by the interaction of a metabolite or other small molecule. Example: CAP has to bind camp before it can bind to DNA. Some bacterial gene regulatory proteins acts as either a activator or a repressor (depending on the precise placement of their DNA-binding sites) Summary of the mechanisms by which specific gene regulatory proteins control gene transcription in procaryotes The operator is located one base pair closer to the promoter Figure 7-38 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-37 Molecular Biology of the Cell ( Garland Science 2008)

17 An activator and a repressor control the lac operon Lac operon encodes proteins to digest lactose. Lac operon is controlled by lac repressor + activator CAP. glu - CAP switches on genes. Utilize alternative sources of carbon (lactose). lac - lac repressor bound and the operon is shut off. Figure 7-39 Molecular Biology of the Cell ( Garland Science 2008) Control of Gene Expression (Eucaryotic cell) Regulatory DNA sequences Nucleosome structure Figure 7-44 Molecular Biology of the Cell ( Garland Science 2008)

18 Eucaryotic transcription regulator control gene expression from a distance Models for Action at a distance Loop out : the enhancer + the promoter DNA acts as a tether. Mediators: serves to link the gene regulatory proteins to the RNApII and GTFs. It was surprising to biologists when, in 1979, it was discovered that these activator proteins could be bound thousands of nucleotide pairs away from the promoter Activator: Aid the assembly of the GTFs and RNApII. Enhancers: DNA sites to which the activators bound. A eucaryotic gene control region consists of a promoter plus regulatory DNA sequences Action mechanism of Activators and Repressors Activators : Assembly of the GTFs and RNA pol II Repressor: Assembly of the GTFs and RNA pol II Tx Activator & Repressor: Attract proteins that modulate chromatin structure. Affect the accessibility of the promoter to the GTFs and RNApII. Gene looping Spacer DNA provides the flexibility needed for efficient DNA looping. Gene regulatory sequence Allows the genes of an organism to be turned on of off individually. Of the roughly 25,000 human genes, an estimated 8% (~2,000 genes) encode gene regulatory proteins.

19 Enhancers The DNA sites to which eucaryotic gene activator proteins bind. Their presence enhanced the rate of transcription initiation. The simplest gene activator proteins Two distinct domains (DNA binding domain + activation domain). A chimeric protein Eucaryotic gene activator proteins promote the assembly of RNA polymerase and the general transcription factors at the startpoint of transcription Figure 7-45a Molecular Biology of the Cell ( Garland Science 2008) Packing of promoter DNA into nucleosomes affects initiation of transcription Nucleosomes can inhibit the initiation of transcription probably because they physically block the assembly of the general transcription factors, RNA polymerase on the promoter In eucaryotic cells, activator and repressor exploit chromatin structure to help turn genes on and off. Figure 7-45b Molecular Biology of the Cell ( Garland Science 2008)

20 1. Chromatin remodeling complex Eucaryotic gene activator proteins also modify local chromatin structure Change the structure of nucleosomes. ATP hydrolysis Accessibility 2. Histone modifying proteins Histone acetyltransferase (HAT) (Gene activator) Acetylation to K of histone; Accessibility Assembly of the GTFs and RNA pol II (Acetyl groups themselves are recognized by proteins that promote transcription). Histone deacetylase (HDAC) (Gene repressor) Deacetylation; Accessibility Figure 7-46 Molecular Biology of the Cell ( Garland Science 2008) General idea The GTFs, mediator, and RNA polymerase seem unable on their own to assemble on a promoter that is packed in standard nucleosomes. Such packing may have evolved to prevent leaky transcription. Four of the most important ways of locally altering chromatin Nucleosome remodeling Nucleosome removal Nucleosome replacement Histone modification Writing and reading the histone code during transcription Writing and reading the histone code during transcription initiation (the human interferon gene promoter) 1. Acetylation on H3K9 and H4K8 Figure 7-47 (part 1 of 4) Molecular Biology of the Cell ( Garland Science 2008)

21 2. Phosphorylation on H3S10 3. Acetylation on H3K14 Figure 7-47 (part 2 of 4) Molecular Biology of the Cell ( Garland Science 2008) Figure 7-47 (part 3 of 4) Molecular Biology of the Cell ( Garland Science 2008) 4. A specific histone code (H3K9ac + H4K8ac + H3S10ph + H3K14ac) recruits chromatin remodeling complex (SWI/SNF) and TFIID. Conclusion: The writing is sequential, with each histone modification depending on a prior modification and the resultant histone code is used to initiate transcription. An order of events leading to transcription initiation of a specific gene. Does histone modification always precede chromatin remodeling? Does mediator enter before or after RNA polymerase? Different for different genes. Figure 7-47 (part 4 of 4) Molecular Biology of the Cell ( Garland Science 2008)

22 Gene activator proteins work synergistically Gene activator proteins often exhibit transcriptional synergy, where several activator proteins working together produce a transcription rate that is much higher than that of the sum of the activators working alone. Figure 7-49 Molecular Biology of the Cell ( Garland Science 2008) Transcription Synergy Synergistic effect vs. Additive effect Eucaryotic gene repressor proteins can inhibit transcription in various ways Competitive DNA binding Masking the activation surface Direct interaction with the general transcription factors Recruitment of chromatin remodeling complexes Recruitment of histone deacetylase Recruitment of histone methyl transferase Figure 7-48 Molecular Biology of the Cell ( Garland Science 2008)

23 Figure 7-50a Molecular Biology of the Cell ( Garland Science 2008) Figure 7-50b Molecular Biology of the Cell ( Garland Science 2008) Figure 7-50c Molecular Biology of the Cell ( Garland Science 2008) Figure 7-50d Molecular Biology of the Cell ( Garland Science 2008)

24 Figure 7-50e Molecular Biology of the Cell ( Garland Science 2008) Figure 7-50f Molecular Biology of the Cell ( Garland Science 2008) Eucaryotic gene regulatory proteins often assemble into complexes on DNA The Molecular Mechanism That Create Specialised Cell Type Developmental Biology Both the red and the green proteins are shared by both activating and repressing complexes. Proteins that do not themselves bind DNA : co-activators or co-repressors. Some have no intrinsic activity themselves but simply serve as a scaffolding to attract those. Figure 7-51 Molecular Biology of the Cell ( Garland Science 2008)

25 Eucaryotic genes are regulated by combinations of proteins Once a cell becomes committed to differentiate into a specific cell type, the choice of fate is generally maintained through many subsequent cell generations. Changes in gene expression involved in the choice must be remembered. cell memory cf. Transient regulation of gene expression (ex, tryptophan repressor). The expression of different genes can be coordinated by a single protein Switch on/off bac cells euk cells Individually combinatorially operon? A single gene regulatory protein decisive Switching any particular gene on or off. Completing the combination to activate or repress. Complete the combination for several different genes. In particular, a eucaryotic cell uses a committee of regulatory proteins to control each of its genes. How can it rapidly and decisively switch whole groups of genes on or off?

26 Glucocorticoid (GC) - GC is a class of steroid hormones that bind to the glucocorticoid receptor. - GC is present in almost every vertebrate animal cell. - Glucocorticoid (glucose + cortex + steroid) : its metabolism regulation of glucose, its synthesis in the adrenal cortex, and its steroidal structure. - GC turns immune activity (inflammation) down. - GCs are therefore used in medicine to treat diseases caused by an overactive immune system, such as allergies ( ), asthma ( ), autoimmune diseases ( ), and sepsis ( ). - Cortisol is the most important human glucocorticoid. - Various synthetic glucocorticoids to treat glucocorticoid deficiency or to suppress the immune system. Figure 7-74 Molecular Biology of the Cell ( Garland Science 2008) Combinational control can create different cell types The fibroblast have already accumulated all of the other necessary gene regulatory proteins required for the combinatorial control of the muscle-specific genes, and that addition of MyoD completes the unique combination that directs the cells to become muscle. A mammalian skeletal muscle cell An extremely large cell. Formed by the fusion of many myoblast. Genes are all switched on coordinately as the myoblasts begin to fuse. Actin, myosin, receptor proteins, ion channel proteins.

27 How do cells ensure that daughter cells remember what kind of cells they are supposed to be? myod Key gene regulatory protein. Coordinates the gene expression. Crucial for muscle cell differentiation. Binds to their regulatory regions. Activates the tx of the genes that code for the muscle-specific proteins. Once a cell has become differentiated... Generally remain differentiated. All its progeny cells will be of that same cell type. Remembered. Stable patterns of gene expression can be transmitted to daughter cells Converts nonmuscle cells to myoblasts (fibroblast to myoblast) Activates the changes in gene expression typical of differentiating muscle cells. 1. Positive feedback loop A key gene regulatory protein activates transcription of its own gene. MyoD 2. Condensed chromatin structure States of chromatin structure can be inherited. A cluster of chromatin proteins bound to the DNA are transferred (epigenetic regulation). X chromosome inactivation.

28 3. DNA methylation DNA methylation on Cytosine bases (Covalent modification). Figure 2.2 Handbook of epigenetic ( Elsevier Inc. 2011) Figure 7-79 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-80 Molecular Biology of the Cell ( Garland Science 2008) DNA methylation DNA methylation on Cytosine bases (Covalent modification). Generally turns off genes by attracting proteins that block gene expression. Passed on to progeny cells by an enzyme that copies the pattern to the daughter DNA strand immediately after replication. *Epigenetic inheritance : transmits information from parent to daughter cell without altering the actual nucleotide sequence of DNA (such as positive feedback, certain forms of condensed chromatin, and DNA methylation) Figure 7-81 Molecular Biology of the Cell ( Garland Science 2008)

29 The formation of an entire organ can be triggered by a single gene regulatory protein Figure 7-77b Molecular Biology of the Cell ( Garland Science 2008) Figure 7-77a Molecular Biology of the Cell ( Garland Science 2008) A single gene regulatory protein (Ey in flies, Pax-6 in vertebrates) Trigger the formation of not just a single cell type but a whole organ the eye (composed of different types of cells all properly organized in three dimensional space). Turns on a cascade of gene regulatory proteins. Forms an organized group of many different types of cells. How does the Ey protein coordinate the specification of each cell in the eye? An actively studied topic in developmental biology. Ey directly controls the expression of many genes by binding to DNA sequences in their regulatory regions. Some of the genes controlled by Ey encode additional transcription regulators that control the expression of other genes. Some of these regulators act back on Ey itself to create a positive feedback loop that ensures the continued production of the Ey protein. Therefore, the action of just one transcription regulator can produce a cascade of regulators whose combined actions lead to the formation of an organized group of many different types of cells.

30 Post-transcriptional Controls Operate after RNA polymerase has bound to a gene s promoter and started to synthesize RNA. Alternative splicing. More examples Riboswitch; UTR of mrna; mirna; sirna Figure 7-92 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-93a Molecular Biology of the Cell ( Garland Science 2008) Figure 7-93b Molecular Biology of the Cell ( Garland Science 2008)

31 Riboswitches Riboswitches provide an economical solution to gene regulation Complex folded RNA domains. Serve as receptors for specific metabolites. Found in non-coding portion of various mrnas. Control gene expression by allosteric structural changes (metabolite binding). A double-stranded structure that forces the polymerase to terminate transcription. Bypass the need for regulatory proteins altogether (Economical examples of gene control). Robust genetic elements in many organisms. Figure 7-93c Molecular Biology of the Cell ( Garland Science 2008) The untranslated region (UTR) of mrnas can control their translation Sequence-specific RNA-binding proteins Gene expression can be controlled by regulating translation initiation (Bacteria) Sequence-specific RNA-binding proteins. Thermosensor RNA sequences. Riboswitches. Antisense RNA. Figure 7-106a Molecular Biology of the Cell ( Garland Science 2008)

32 Thermosensor RNA sequences Riboswitches Figure 7-106b Molecular Biology of the Cell ( Garland Science 2008) Figure 7-106c Molecular Biology of the Cell ( Garland Science 2008) Antisense RNA microrna (mirna) Small regulatory RNAs control the expression of thousands of animal and plant genes Noncoding RNAs transcribed by Pol II. More than 400 different mirnas in human. Regulate at least one-third of all protein-coding genes. Base-paring with specific mrna s and controlling their stability and translation. Form an RNA-induced silencing complex (RISC). Target mrna is destroyed immediately by a nuclease present within the RISC. Figure 7-106d Molecular Biology of the Cell ( Garland Science 2008)

33 Plants Animals Figure Molecular Biology of the Cell ( Garland Science 2008) Figure Molecular Biology of the Cell ( Garland Science 2008) What features of mirna make them especially useful regulators of gene expression? First, a single mirna can regulate a whole set of different mrna so long as the mrnas carry a common sequence (5 and 3 UTRs; some individual mirnas control hundreds of different mrnas in human). mrna decapping enzyme Dcp1 Argonaute Merge Second, a gene that encodes an mirna occupies relatively little space in the genome compared with one that encodes a transcription regulator (Small size). Two proteins co-localized to P-bodies Figure Molecular Biology of the Cell ( Garland Science 2008)

34 RNA interference is a cell defines mechanism RNA interference (RNAi) A cell defense mechanism. Orchestrate the destruction of foreign double-stranded RNA (From viruses or transposable genetic elements; Targeted RNA degradation mechanism). RNAi response resembles human immune system. sirna for destruction of target RNA molecules RITS (RNA-induced transcriptional silencing) complex attracts proteins and direct the heterochromatin to prevent further transcriptional initiation Figure Molecular Biology of the Cell ( Garland Science 2008) Action mechanism by which sirna destroy foreign RNAs. Foreign RNA attracts Dicer (a protein complex with nuclease). Dicer cleaves the double-stranded RNA into short fragments (about 23 nt); small interfering RNAs (sirnas). sirnas (short, double-stranded RNAs) are then incorporated into RISCs. RISC discard one strand and uses the remaining single-stranded RNA to locate a complementary foreign RNA molecule. This targeted RNA molecule is then rapidly degraded. Scientists can use RNA interference to turn off genes RNAi has become powerful experimental tool to inactivate almost any gene in cells (Chapter 10). RNAi as a powerful new approach for treating human disease through the regulation of gene expression (sirna for medical promise). RNAi expands our understanding of the types of regulatory networks to specify the development of complex organisms, including ourselves.