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Chapter 18: Regulation of Gene Expression 1. Gene Regulation in Bacteria 2. Gene Regulation in Eukaryotes 3. Gene Regulation in Development 4. Gene Regulation & Cancer Gene Regulation Gene regulation refers to all aspects of controlling the levels and/or activities of specific gene products. • the gene product is either a protein or an RNA molecule • regulation can occur at any stage of gene expression which involves • accessibility of the gene itself (chromatin structure) • transcription & translation (if gene encodes protein) • modification of the gene product Transcription Factors Transcription factors are proteins that either help activate or inhibit transcription. Many transcription factors bind to specific DNA sequences in the regulatory regions of genes. Activation domain DNA-binding domain DNA • DNA-binding transcription factors have a DNA-binding domain and one or more activation domains that mediate effects on transcription 1. Gene Regulation in Bacteria Chapter Reading – pp. 361-364 Bacterial Gene Regulation Gene regulation in bacteria is generally accomplished at the levels of transcription and post-translational modification of protein activity. Bacterial genes are commonly organized in multi-gene structures called operons: • multiple gene coding regions organized in sequence under control of a single promoter • genes in the operon are part of same metabolic pathway • operons are typically inducible or repressible Regulation of Tryptophan Production Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Enzyme 2 Regulation of gene expression trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity • enzymes involved in tryptophan synthesis are part of a single operon (b) Regulation of enzyme production • regulation involves transcription & posttranslational modification (feedback inhibition) trp operon Promoter Promoter Genes of operon DNA trpE trpR trpD trpC trpB trpA C B A Operator Regulatory gene 3 RNA polymerase Start codon Stop codon mRNA 5 mRNA 5 E Protein Inactive repressor D Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on The trp Operon DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off • trp repressor is inactive unless bound to tryptophan • low tryptophan = ON • high tryptophan = OFF repressible operon Regulatory gene DNA Promoter The lac Operon Operator lacI lacZ No RNA made 3 mRNA RNA polymerase 5 • low allolactose = OFF Active repressor Protein • lac repressor is active unless bound to allolactose (a) Lactose absent, repressor active, operon off • high allolactose = ON lac operon DNA lacI lacZ lacY lacA RNA polymerase 3 mRNA 5 mRNA 5 -Galactosidase Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on Permease Transacetylase inducible operon …more on the lac Operon Promoter DNA lacI lacZ CAP-binding site When ON the lac operon is on “low” by default If glucose (preferred sugar) is unavailable, lac operon is “turned up” due to CAP activation • cAMP is produced if glucose is low • cAMP binds and activates CAP • active CAP binds CAP site increasing Tx cAMP Operator RNA polymerase Active binds and transcribes CAP Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Promoter DNA lacI CAP-binding site lacZ Operator RNA polymerase less likely to bind Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized 2. Gene Regulation in Eukaryotes Chapter Reading – pp. 365-376 Overview of Eukaryotic Gene Regulation Eukaryotic genes generally have the following: • a single coding region consisting of exons & introns • a single promoter • multiple proximal and distal control sequences • distal control sequences can be 1000s of base pairs away Eukaryotic gene regulation is dependent on chromatin structure in addition all stages between transcription initiation and the production of a functional gene product. Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation DNA Gene available for transcription Gene Stages of Gene Regulation Chromatin structure* • controls access to genes Transcription RNA Exon Primary transcript Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA • key stage of gene regulation RNA processing* • splicing of the RNA transcript Translation Polypeptide Protein processing, such as cleavage and chemical modification Degradation of protein Transcription RNA stability Translation of mRNA Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Post-translation modifications *relevant to eukaryotes only Chromatin Structure Chromatin structure is regulated through modifications of either the DNA itself or the histone proteins associated with the DNA: DNA modifications • addition of methyl (CH3) groups to cytosines • results in more compact, less accessible chromatin • responsible for X-inactivation, genomic imprinting Histone modifications • addition of acetyl groups (“opens” chromatin) • addition of CH3 (“closed”) or PO4 (“open”) groups Histones & Chromatin Structure Histone tails Amino acids available for chemical modification DNA double helix Nucleosome (end view) (a) Histone tails protrude outward from a nucleosome Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription DNA is wrapped around histone cores in structures called nucleosomes. • tails of histone proteins in nucleosomes can have acetyl, methyl or phosphate groups added to induce a more “open” or “closed” chromatin structure Proximal & Distal Regulation Enhancer (distal control elements) Proximal control elements Transcription start site Exon DNA Upstream Distal elements interact with promoter due to bending of DNA. Intron Exon Intron Downstream Poly-A signal Intron Exon Exon Cleaved 3 end of primary RNA processing transcript Promoter Primary RNA transcript (pre-mRNA) Poly-A Transcription signal sequence termination region Intron Exon Transcription Exon 5 Intron RNA Coding segment mRNA G P P 5 Cap AAA AAA P 5 UTR Start codon Stop codon 3 UTR 3 Poly-A tail • control elements bind specific transcription factors • can be located near the promoter (proximal) or very far from the promoter (distal) Promoter Activators DNA Enhancer Distal control element Current Model of Eukaryotic Transcription Initiation Gene TATA box General transcription factors DNAbending protein Group of mediator proteins RNA polymerase II Involves specific transcription factors as well as general transcription factors and other proteins involved in all transcription Initiation. RNA polymerase II Transcription initiation complex RNA synthesis Differential Gene Expression Different genes are expressed in different cell types due to: Enhancer Control elements Promoter Albumin gene Crystallin gene LENS CELL NUCLEUS LIVER CELL NUCLEUS Available activators Available activators Albumin gene not expressed • differences in transcription factors Albumin gene expressed • differences in chromatin structure Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell Regulatory roles of non-coding RNA Spliceosomes • contain snRNA molecules that direct the process of splicing introns from primary RNA transcripts MicroRNAs (miRNA) • complex with specific proteins to facilitate destruction of specific mRNA molecules that contain sequences complementary to miRNA sequence • target chromatin modification to the centromeres of chromosomes resulting in highly condensed heterochromatin in the centromeres • protection from infection by RNA viruses Alternative Splicing of RNA Exons DNA 1 3 2 4 5 Troponin T gene Primary RNA transcript 3 2 1 5 4 RNA splicing mRNA 1 2 3 5 or 1 2 4 5 Hairpin Hydrogen bond miRNA Dicer 5 3 (a) Primary miRNA transcript miRNA miRNAprotein complex miRNA Production mRNA degraded Translation blocked (b) Generation and function of miRNAs Protein Degradation Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein entering a proteasome Protein fragments (peptides) • proteins to be degraded in cells (e.g., cyclins) are “tagged” with a small protein called ubiquitin • ubiquitinated proteins are directed to proteosomes which then degrade them Transcription Chromatin modification • Genes in highly compacted chromatin are generally not transcribed. • Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. • DNA methylation generally reduces transcription. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. • Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Chromatin modification Transcription RNA processing RNA processing • Alternative RNA splicing: Primary RNA transcript mRNA degradation Translation mRNA Protein processing and degradation or Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation. Summary of Eukaryotic Gene Regulation 3. Gene Regulation in Development Chapter Reading – pp. 376-382 Embryonic Development “From fertilization to fully developed organism.” Involves regulation of maternal and embryonic gene expression: 1 mm (a) Fertilized eggs of a frog • maternal genes involved in packaging the egg during oogenesis (egg production) 2 mm (b) Newly hatched tadpole Eye Leg Antenna Wild type Mutant • embryonic genes control development after fertilization Mutations in either maternal or embryonic genes can result in developmental defects Key Events in Animal Development Oogenesis • egg production in the ovary results in essential gene regulatory factors (RNA, protein) being packaged very specifically and unevenly in the developing egg Fertilization • triggers translation of maternal mRNA and rapid series of mitotic nuclear divisions (cleavage) Gastrulation & Induction • cell rearrangement and cell-cell signaling resulting in the differentiation of cells and formation of distinct body structures Early Development (a) Cytoplasmic determinants in the egg (b) Induction by nearby cells Unfertilized egg Sperm Fertilization Cell-cell communication also induces changes in gene expression. Nucleus Molecules of two different cytoplasmic determinants Early embryo (32 cells) NUCLEUS Zygote (fertilized egg) Mitotic cell division Two-celled embryo Egg is packaged unevenly with regulatory factors that are then partitioned into different cells after fertilization. Signal transduction pathway Signal receptor Signaling molecule (inducer) Head Thorax Abdomen (a) Adult Follicle cell 1 Egg developing within ovarian follicle Nucleus Egg 0.5 mm Nurse cell Dorsal BODY AXES Anterior Left Right Posterior 2 Unfertilized egg Depleted nurse cells Fertilization Ventral Early Drosophila Development • maternal genes determine body axes and early pattern formation • embryonic genes eventually take over and determine subsequent morphogenesis Egg shell Laying of egg 3 Fertilized egg Embryonic development 4 Segmented embryo 0.1 mm Body segments Hatching 5 Larval stage (b) Development from egg to larva Bicoid Determines Anterior End The mutant phenotype named “Bicoid” results in larva with 2 posteriors and no anterior (NO head!). • due to a mutation Head in the maternal Bicoid gene • Bicoid mRNA is deposited in the anterior end of all eggs during oogenesis • Bicoid activates anterior gene expression after fertilization Tail T1 T2 T3 A8 A6 A1 A2 A3 A4 A5 Wild-type larva A7 250 m Tail Tail A8 A8 A7 A6 A7 Mutant larva (bicoid) Localization of Bicoid Protein, mRNA RESULTS Bicoid is a Anterior end morphogen of maternal origin 100 m Fertilization, translation of bicoid mRNA Bicoid mRNA in mature unfertilized egg Bicoid mRNA in mature unfertilized egg Bicoid mRNA is expressed into protein after fertilization This results in a Bicoid “morphogen gradient” Bicoid protein in early embryo Bicoid protein in early embryo Nucleus Embryonic precursor cell Master regulatory gene myoD Other muscle-specific genes DNA Myoblast (determined) OFF OFF mRNA OFF MyoD protein (transcription factor) mRNA MyoD Part of a muscle fiber (fully differentiated cell) Specification of vertebrate muscle tissue mRNA Another transcription factor mRNA mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins 4. Gene Regulation & Cancer Chapter Reading – pp. 383-388 Oncogenes Oncogenes are genes with a role in cell cycle progression that have undergone a mutation that contributes to cancer formation (normal version is called a proto-oncogene). • generally due to dominant “gain-of-function” mutations • mutations are of 3 general types: 1) translocation of the gene 2) amplification (duplication) of the gene 3) mutations in the coding or regulatory regions of the gene More on Oncogenes Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene New promoter Normal growthstimulating protein in excess Point mutation: within a control within element the gene Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess Oncogene Hyperactive or degradationresistant protein • mutations that result in excessive expression or function can contribute to cancer MUTATION 1 Growth factor Ras 3 G protein GTP Ras P P P 2 Receptor P P P Hyperactive Ras protein (product of oncogene) issues signals on its own. GTP 4 Protein kinases (phosphorylation cascade) Ras is a G protein that is a proto-oncogene. NUCLEUS 5 Transcription factor (activator) Gain-of-function Ras mutations can trigger “signal-independent” activation of cell cycle. (a) Cell cycle–stimulating pathway DNA Gene expression Protein that stimulates the cell cycle Tumor Suppressor Genes Tumor Suppressor Genes encode gene products that inhibit cell cycle progression. Mutations in tumor suppressor genes are typically recessive “loss-of-function” mutations. • typically requires 2 mutant alleles (recessive) • loss of functional gene product leads to defect in: • inhibiting cell cycle progression • triggering apoptosis • activating DNA repair 2 Protein kinases MUTATION 3 Active form of p53 UV light 1 DNA damage in genome DNA Protein that inhibits the cell cycle (b) Cell cycle–inhibiting pathway Defective or missing transcription factor, such as p53, cannot activate transcription. Cancer Requires Multiple Mutations The “multi-step” or “multi-hit” hypothesis. EFFECTS OF MUTATIONS Protein overexpressed Protein absent Colon Cell cycle overstimulated Increased cell division Cell cycle not inhibited (c) Effects of mutations 1 Loss of tumorsuppressor gene APC (or other) 2 Activation of ras oncogene Colon wall Normal colon epithelial cells 4 Loss of tumorsuppressor gene p53 Small benign growth (polyp) 3 Loss of tumorsuppressor gene DCC Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma) Key Terms for Chapter 18 • nucleosome, euchromatin, heterochromatin • operon, repressor, operator, repressible, inducible • control elements, distal, proximal, enhancer • general vs specific transcription factors • mediator proteins, DNA bending protein Relevant Chapter Questions • miRNA, alternative RNA splicing, Dicer, hairpin • oogenesis, cytoplasmic determinants, induction • morphogenesis, morphogen, morphogen gradient • oncogene, proto-oncogene, tumor suppressor gene • gain-of-function, loss-of-function mutations 1-11