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LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 18 Regulation of Gene Expression Lectures by Erin Barley Kathleen Fitzpatrick © 2011 Pearson Education, Inc. Overview: Conducting the Genetic Orchestra • Prokaryotes and eukaryotes alter gene expression in response to their changing environment • In multicellular eukaryotes, gene expression regulates development and is responsible for cell differentiation • RNA molecules play many roles in regulating gene expression in eukaryotes © 2011 Pearson Education, Inc. Figure 18.1 Concept 18.1: Bacteria often respond to environmental change by regulating transcription • Bacterial cells rely on regulating their gene expression to conserve energy and resources. • Prokaryotes arrange genes that are expressed together and function as a unit in operons. © 2011 Pearson Education, Inc. Figure 18.2 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 (b) Regulation of enzyme production Operons: The Basic Concept • A cluster of functionally related genes can be under coordinated control by a single “on-off switch” • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control © 2011 Pearson Education, Inc. • The operon can be switched off by a protein repressor • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase • The repressor is the product of a separate regulatory gene © 2011 Pearson Education, Inc. • The repressor can be in an active or inactive form, depending on the presence of other molecules • A corepressor is a molecule that cooperates with a repressor protein to switch an operon off • For example, E. coli can synthesize the amino acid tryptophan © 2011 Pearson Education, Inc. • By default the trp operon is on and the genes for tryptophan synthesis are transcribed • When tryptophan is present, it binds to the trp repressor protein, which turns the operon off • The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high © 2011 Pearson Education, Inc. Figure 18.3a trp operon Promoter Promoter Genes of operon DNA trpR Regulatory gene mRNA trpE 3′ Operator RNA Start codon polymerase mRNA 5′ trpD trpC trpB trpA C B A Stop codon 5′ E Protein Inactive repressor D Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on Figure 18.3b-1 DNA mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Figure 18.3b-2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Repressible and Inducible Operons: Two Types of Negative Gene Regulation • A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription • The trp operon is a repressible operon • An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription © 2011 Pearson Education, Inc. • The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose • By itself, the lac repressor is active and switches the lac operon off • A molecule called an inducer inactivates the repressor to turn the lac operon on © 2011 Pearson Education, Inc. Figure 18.4a Regulatory gene DNA Promoter Operator lacI lacZ No RNA made 3′ mRNA 5′ Protein RNA polymerase Active repressor (a) Lactose absent, repressor active, operon off Figure 18.4b lac operon lacI DNA lacZ lacY lacA Permease Transacetylase RNA polymerase 3′ mRNA 5′ mRNA 5′ β-Galactosidase Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on • Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal • Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor © 2011 Pearson Education, Inc. Positive Gene Regulation • Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription • When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP (cAMP) • Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription © 2011 Pearson Education, Inc. • When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate • CAP helps regulate other operons that encode enzymes used in catabolic pathways © 2011 Pearson Education, Inc. Figure 18.5a Promoter DNA lacI lacZ CAP-binding site 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 Figure 18.5b 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 Concept 18.2: Eukaryotic gene expression is regulated at many stages • All organisms must regulate which genes are expressed at any given time • In multicellular organisms regulation of gene expression is essential for cell specialization © 2011 Pearson Education, Inc. Differential Gene Expression • Almost all the cells in an organism are genetically identical • Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages © 2011 Pearson Education, Inc. Figure 18.6a Signal NUCLEUS Chromatin DNA Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM Figure 18.6b CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing, such as cleavage and chemical modification Degradation of protein Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Regulation of Chromatin Structure • Genes within highly packed heterochromatin are usually not expressed • Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression © 2011 Pearson Education, Inc. Histone Modifications • In histone acetylation, acetyl groups are attached to histone tails • This loosens chromatin structure, thereby promoting the initiation of transcription • The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin © 2011 Pearson Education, Inc. Animation: DNA Packing Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 18.7 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 Methylation • DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation • In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development © 2011 Pearson Education, Inc. Regulation of Transcription Initiation • Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery © 2011 Pearson Education, Inc. Organization of a Typical Eukaryotic Gene • Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription © 2011 Pearson Education, Inc. Figure 18.8-1 Enhancer (distal control elements) DNA Upstream Proximal control elements Transcription start site Exon Promoter Intron Exon Poly-A signal Transcription sequence termination region Intron Exon Downstream Figure 18.8-2 Enhancer (distal control elements) DNA Upstream Proximal control elements Transcription start site Exon Intron Promoter Primary RNA transcript 5′ (pre-mRNA) Exon Poly-A signal Transcription sequence termination region Intron Exon Downstream Poly-A signal Intron Exon Cleaved 3′ end of primary transcript Transcription Exon Intron Exon Figure 18.8-3 Enhancer (distal control elements) Proximal control elements Transcription start site Exon DNA Upstream Intron Exon Intron Downstream Poly-A signal Intron Exon Exon Cleaved 3′ end of primary RNA processing transcript Promoter Transcription Exon Primary RNA transcript 5′ (pre-mRNA) Poly-A signal Transcription sequence termination region Intron Exon Intron RNA Coding segment mRNA G P AAA ⋅⋅⋅ AAA P P 5′ Cap 5′ UTR Start Stop codon codon 3′ UTR Poly-A tail 3′ The Roles of Transcription Factors • To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors • Proximal control elements are located close to the promoter • Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron © 2011 Pearson Education, Inc. • An activator is a protein that binds to an enhancer and stimulates transcription of a gene • Activators have two domains, one that binds DNA and a second that activates transcription • Bound activators facilitate a sequence of proteinprotein interactions that result in transcription of a given gene © 2011 Pearson Education, Inc. Animation: Initiation of Transcription Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 18.9 Activation domain DNA-binding domain DNA • Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription © 2011 Pearson Education, Inc. Figure 18.10-1 Activators Promoter DNA Enhancer Distal control element TATA box Gene Figure 18.10-2 Promoter Activators DNA Enhancer Distal control element Gene TATA box General transcription factors DNAbending protein Group of mediator proteins Figure 18.10-3 Promoter Activators DNA Enhancer Distal control element Gene TATA box General transcription factors DNAbending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis Figure 18.11a Enhancer Control elements Promoter LIVER CELL NUCLEUS Albumin gene Crystallin gene Available activators Albumin gene expressed Crystallin gene not expressed (a) Liver cell Figure 18.11b Enhancer Control elements Promoter LENS CELL NUCLEUS Albumin gene Crystallin gene Available activators Albumin gene not expressed Crystallin gene expressed (b) Lens cell Coordinately Controlled Genes in Eukaryotes • Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elements • These genes can be scattered over different chromosomes, but each has the same combination of control elements • Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes © 2011 Pearson Education, Inc. RNA Processing • In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns © 2011 Pearson Education, Inc. Animation: RNA Processing Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 18.13 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 mRNA Degradation • The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis • Eukaryotic mRNA is more long lived than prokaryotic mRNA • Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3′ end of the molecule © 2011 Pearson Education, Inc. Animation: mRNA Degradation Right-click slide / select “Play” © 2011 Pearson Education, Inc. Animation: Blocking Translation Right-click slide / select “Play” © 2011 Pearson Education, Inc. Protein Processing and Degradation • After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control • Proteasomes are giant protein complexes that bind protein molecules and degrade them © 2011 Pearson Education, Inc. Animation: Protein Processing Right-click slide / select “Play” © 2011 Pearson Education, Inc. Animation: Protein Degradation Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 18.14 Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Proteasome and ubiquitin to be recycled Protein entering a proteasome Protein fragments (peptides) Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression • Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA • Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration © 2011 Pearson Education, Inc. Effects on mRNAs by MicroRNAs and Small Interfering RNAs • MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation © 2011 Pearson Education, Inc. Figure 18.15 Hairpin Hydrogen bond miRNA Dicer 5′ 3′ (a) Primary miRNA transcript miRNA miRNAprotein complex mRNA degraded Translation blocked (b) Generation and function of miRNAs • The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) • RNAi is caused by small interfering RNAs (siRNAs) • In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome © 2011 Pearson Education, Inc. Concept 18.4: A program of differential gene expression leads to the different cell types in a multicellular organism • During embryonic development, a fertilized egg gives rise to many different cell types • Cell types are organized successively into tissues, organs, organ systems, and the whole organism • Gene expression orchestrates development. © 2011 Pearson Education, Inc. Figure 18.16 1 mm (a) Fertilized eggs of a frog 2 mm (b) Newly hatched tadpole • Cell differentiation is the process by which cells become specialized in structure and function • The physical processes that give an organism its shape constitute morphogenesis • Differential gene expression results from genes being regulated differently in each cell type • Materials in the egg can set up gene regulation that is carried out as cells divide © 2011 Pearson Education, Inc. • The other important source of developmental information is the environment around the cell, especially signals from nearby embryonic cells • In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells • Thus, interactions between cells induce differentiation of specialized cell types © 2011 Pearson Education, Inc. Animation: Cell Signaling Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 18.17b (b) Induction by nearby cells Early embryo (32 cells) NUCLEUS Signal transduction pathway Signal receptor Signaling molecule (inducer) Sequential Regulation of Gene Expression During Cellular Differentiation • Determination commits a cell to its final fate • Determination precedes differentiation • Cell differentiation is marked by the production of tissue-specific proteins © 2011 Pearson Education, Inc. Figure 18.18-3 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) mRNA Another transcription factor mRNA mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins Concept 8.4: Enzymes speed up metabolic reactions by lowering energy barriers • A catalyst is a chemical agent that speeds up a reaction without being consumed by the reaction • An enzyme is a catalytic protein • Hydrolysis of sucrose by the enzyme sucrase is an example of an enzyme-catalyzed reaction © 2011 Pearson Education, Inc. Figure 8.UN02 Sucrase Sucrose (C12H22O11) Glucose (C6H12O6) Fructose (C6H12O6) The Activation Energy Barrier • Every chemical reaction between molecules involves bond breaking and bond forming • The initial energy needed to start a chemical reaction is called the free energy of activation, or activation energy (EA) • Activation energy is often supplied in the form of thermal energy that the reactant molecules absorb from their surroundings © 2011 Pearson Education, Inc. Figure 8.12 A B C D Free energy Transition state A B C D EA Reactants A B ∆G < O C D Products Progress of the reaction How Enzymes Lower the EA Barrier • Enzymes catalyze reactions by lowering the EA barrier • Enzymes do not affect the change in free energy (∆G); instead, they hasten reactions that would occur eventually © 2011 Pearson Education, Inc. Animation: How Enzymes Work Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 8.13 Free energy Course of reaction without enzyme EA without enzyme EA with enzyme is lower Reactants ∆G is unaffected by enzyme Course of reaction with enzyme Products Progress of the reaction Substrate Specificity of Enzymes • The reactant that an enzyme acts on is called the enzyme’s substrate • The enzyme binds to its substrate, forming an enzyme-substrate complex • The active site is the region on the enzyme where the substrate binds • Induced fit of a substrate brings chemical groups of the active site into positions that enhance their ability to catalyze the reaction © 2011 Pearson Education, Inc. Figure 8.14 Substrate Active site Enzyme (a) Enzyme-substrate complex (b) Catalysis in the Enzyme’s Active Site • In an enzymatic reaction, the substrate binds to the active site of the enzyme • The active site can lower an EA barrier by – – – – Orienting substrates correctly Straining substrate bonds Providing a favorable microenvironment Covalently bonding to the substrate © 2011 Pearson Education, Inc. Figure 8.15-1 1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex Active site Enzyme Figure 8.15-2 1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex 3 Active site can lower EA and speed up a reaction. Active site Enzyme 4 Substrates are converted to products. Figure 8.15-3 1 Substrates enter active site. 2 Substrates are held in active site by weak interactions. Substrates Enzyme-substrate complex 3 Active site can lower EA and speed up a reaction. 6 Active site is available for two new substrate molecules. Enzyme 5 Products are released. 4 Substrates are converted to products. Products Effects of Local Conditions on Enzyme Activity • An enzyme’s activity can be affected by – General environmental factors, such as temperature and pH – Chemicals that specifically influence the enzyme • Enzymes are environment specific- some function well in high temperatures and/or extreme pH. © 2011 Pearson Education, Inc. Effects of Temperature and pH • Each enzyme has an optimal temperature in which it can function • Each enzyme has an optimal pH in which it can function • Optimal conditions favor the most active shape for the enzyme molecule © 2011 Pearson Education, Inc. Figure 8.16 Rate of reaction Optimal temperature for Optimal temperature for typical human enzyme (37°C) enzyme of thermophilic (heat-tolerant) bacteria (77°C) 60 80 Temperature (°C) (a) Optimal temperature for two enzymes 0 20 40 Rate of reaction Optimal pH for pepsin (stomach enzyme) 0 5 pH (b) Optimal pH for two enzymes 1 2 3 4 120 100 Optimal pH for trypsin (intestinal enzyme) 6 7 8 9 10 Figure 8.16a Rate of reaction Optimal temperature for Optimal temperature for typical human enzyme (37°C) enzyme of thermophilic (heat-tolerant) bacteria (77°C) 60 80 Temperature (°C) (a) Optimal temperature for two enzymes 0 20 40 100 120 Rate of reaction Figure 8.16b Optimal pH for pepsin (stomach enzyme) 0 5 pH (b) Optimal pH for two enzymes 1 2 3 4 Optimal pH for trypsin (intestinal enzyme) 6 7 8 9 10 Cofactors • Cofactors are nonprotein enzyme helpers • Cofactors may be inorganic (such as a metal in ionic form) or organic • An organic cofactor is called a coenzyme • Coenzymes include vitamins © 2011 Pearson Education, Inc. Enzyme Inhibitors • Competitive inhibitors bind to the active site of an enzyme, competing with the substrate • Noncompetitive (allosteric) inhibitors bind to another part of an enzyme, causing the enzyme to change shape and making the active site less effective • Examples of inhibitors include toxins, poisons, pesticides, and antibiotics © 2011 Pearson Education, Inc. Figure 8.17 (a) Normal binding (b) Competitive inhibition (c) Noncompetitive inhibition Substrate Active site Competitive inhibitor Enzyme Noncompetitive inhibitor The Evolution of Enzymes • Enzymes are proteins encoded by genes • Changes (mutations) in genes lead to changes in amino acid composition of an enzyme • Altered amino acids in enzymes may alter their substrate specificity • Under new environmental conditions a novel form of an enzyme might be favored © 2011 Pearson Education, Inc. Concept 8.5: Regulation of enzyme activity helps control metabolism • Chemical chaos would result if a cell’s metabolic pathways were not tightly regulated • A cell does this by switching on or off the genes that encode specific enzymes or by regulating the activity of enzymes © 2011 Pearson Education, Inc. Allosteric Regulation of Enzymes • Allosteric regulation may either inhibit or stimulate an enzyme’s activity • Allosteric regulation occurs when a regulatory molecule binds to a protein at one site and affects the protein’s function at another site © 2011 Pearson Education, Inc. Allosteric Activation and Inhibition • Most allosterically regulated enzymes are made from polypeptide subunits • Each enzyme has active and inactive forms • The binding of an activator stabilizes the active form of the enzyme • The binding of an inhibitor stabilizes the inactive form of the enzyme © 2011 Pearson Education, Inc. Figure 8.19 (b) Cooperativity: another type of allosteric activation (a) Allosteric activators and inhibitors Allosteric enzyme with four subunits Active site (one of four) Regulatory site (one of four) Substrate Activator Inactive form Stabilized active form Active form Oscillation Nonfunctional active site Inactive form Inhibitor Stabilized inactive form Stabilized active form Figure 8.19a (a) Allosteric activators and inhibitors Allosteric enzyme with four subunits Active site (one of four) Regulatory site (one of four) Activator Active form Stabilized active form Oscillation Nonfunctional active site Inactive form Inhibitor Stabilized inactive form Figure 8.19b (b) Cooperativity: another type of allosteric activation Substrate Inactive form Stabilized active form • Cooperativity is a form of allosteric regulation that can amplify enzyme activity • One substrate molecule primes an enzyme to act on additional substrate molecules more readily • Cooperativity is allosteric because binding by a substrate to one active site affects catalysis in a different active site © 2011 Pearson Education, Inc. Feedback Inhibition • In feedback inhibition, the end product of a metabolic pathway shuts down the pathway • Feedback inhibition prevents a cell from wasting chemical resources by synthesizing more product than is needed © 2011 Pearson Education, Inc. Figure 8.21 Active site available Isoleucine used up by cell Active site of Feedback enzyme 1 is inhibition no longer able to catalyze the conversion of threonine to intermediate A; pathway is switched off. Isoleucine binds to allosteric site. Initial substrate (threonine) Threonine in active site Enzyme 1 (threonine deaminase) Intermediate A Enzyme 2 Intermediate B Enzyme 3 Intermediate C Enzyme 4 Intermediate D Enzyme 5 End product (isoleucine)