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Eldra Solomon Linda Berg Diana W. Martin www.cengage.com/biology/solomon Chapter 14 Gene Regulation Albia Dugger • Miami Dade College Gene Regulation • Cells differ because gene expression is regulated, and only certain subsets of the total genetic information are expressed in any given cell • Gene expression involves three basic steps, each of which is regulated in many ways: • Transcribing the gene to form messenger RNA (mRNA) • Translating mRNA into protein • Activating the protein Control Mechanisms • Mechanisms that regulate gene expression include: • control of the amount of mRNA transcribed • rate of translation of mRNA • activity of the protein product • Control mechanisms use various signals, some originating within the cell and others coming from other cells or from the environment 14.1 GENE REGULATION IN BACTERIA AND EUKARYOTES LEARNING OBJECTIVE: • Explain why bacterial and eukaryotic cells have different mechanisms of gene regulation Bacterial Gene Regulation • The main requirement of bacterial gene regulation is production of enzymes and other proteins when needed • Transcriptional-level control is the most efficient mechanism – bacteria rarely regulate enzyme levels by degrading proteins • Related genes are organized into groups that are rapidly turned on and off as units Eukaryotic Gene Regulation • Because a single gene is regulated in different ways in different types of cells, eukaryotic gene regulation is complex • Transcriptional-level control predominates, but control at other levels of gene expression is also very important, especially in multicellular organisms • In many instances, pre-formed enzymes and other proteins are rapidly converted from an inactive to an active state • In multicellular organisms, each type of cell has certain genes that are active and other genes that may never be used KEY CONCEPTS 14.1 • Cells regulate which genes will be expressed and when • Gene regulation in bacteria is primarily in response to stimuli in the environment; gene regulation in eukaryotes helps to maintain homeostasis 14.2 GENE REGULATION IN BACTERIA LEARNING OBJECTIVES: • Define operon and explain the functions of the operator and promoter regions • Distinguish among inducible, repressible, and constitutive genes • Differentiate between positive and negative control, and show how both types of control operate in regulating the lac operon • Describe the types of posttranscriptional control in bacteria Bacterial Gene Expression • The E. coli bacterium has some genes that encode proteins that are always needed (constitutive genes), and some that encode proteins that are needed only in certain conditions • Example: • E. coli living in the colon of a calf need enzymes that digest the milk sugar lactose • E. coli living in the colon of an adult cow are not exposed to milk and do not need lactose-digesting enzymes Enzyme Activity • Cell metabolic activity is controlled in two ways: • By regulating the activity of certain enzymes (how effectively an enzyme molecule works) • By regulating the number of enzyme molecules present in each cell • Bacteria respond rapidly to changing environmental conditions because functionally related genes are regulated together in gene complexes called operons Operons in Bacteria • The DNA coding sequences for all three enzymes needed to digest lactose are linked as a unit on bacterial DNA and are controlled by a common mechanism • Each enzyme-coding sequence is a structural gene • operon • A gene complex consisting of a group of structural genes with related functions plus the closely linked DNA sequences responsible for controlling them The lac Operon • The structural genes of the lactose operon (lac operon)— lacZ, lacY, and lacA—code for three enzymes • RNA polymerase binds to a promoter region upstream from the coding sequences • A single mRNA molecule is transcribed that contains the coding information for all three enzymes • mRNA synthesis is controlled by a single molecular “switch,” the operator The lac Operator • The operator is a sequence of bases upstream from the first structural gene in the operon • In the absence of lactose, a repressor protein (lactose repressor) binds tightly to the operator • RNA polymerase binds to the promoter but is blocked from transcribing the protein-coding genes of the lac operon The Lactose Repressor • Lactose repressor protein is encoded by a repressor gene located upstream from the promoter site • The repressor gene is a structural gene that is constitutively expressed (constantly transcribed) • In the absence of lactose, the repressor protein binds specifically to the lac operator sequence, and transcription of the lac operon is suppressed Turing On Transcription • Lactose “turns on” (induces ) transcription of the lac operon • The lactose repressor protein contains a second functional region (an allosteric site) separate from its DNA-binding site • In the presence of lactose, the allosteric site binds to the inducer (allolactose) which inactivates the repressor protein • RNA polymerase is able to actively transcribes the structural genes of the operon The lac Operon lac operon Repressor gene Promoter Operator lacZ lacY lacA DNA Transcription Repressor protein mRNA Precursor of repressor protein Ribosome Translation (a) Lactose absent. In the absence of lactose, a repressor protein, encoded by an adjacent gene, binds to a region known as the operator, thereby blocking transcription of the structural genes. Fig. 14-2a, p. 310 lac operon Repressor gene Promoter Operator lacZ lacY lacA DNA Transcription Repressor protein mRNA Precursor of repressor protein Ribosome Translation (a) Lactose absent. In the absence of lactose, a repressor protein, encoded by an adjacent gene, binds to a region known as the operator, thereby blocking transcription of the structural genes. Stepped Art Fig. 14-2a, p. 310 lac operon Repressor gene mRNA Promoter OperatorlacZ lacY lacA RNA polymerase Transcription mRNA Inducer (allolactose) Repressor protein (inactive) Translation Transacetylase Lactose permease β -galactosidase Enzymes for lactose metabolism (b) Lactose present. When lactose is present, it is converted to allolactose, which binds to the repressor at an allosteric site, altering the structure of the protein so it no longer binds to the operator. As a result, RNA polymerase is able to transcribe the structural genes. Fig. 14-2b, p. 310 lac operon Repressor gene mRNA Promoter Operator lacZ lacY lacA RNA polymerase Transcription mRNA Inducer (allolactose) Repressor protein (inactive) Translation Transacetylase Lactose permease β -galactosidase Enzymes for lactose metabolism (b) Lactose present. When lactose is present, it is converted to allolactose, which binds to the repressor at an allosteric site, altering the structure of the protein so it no longer binds to the operator. As a result, RNA polymerase is able to Stepped Art transcribe the structural genes. Fig. 14-2b, p. 310 Inducible Genes • The lac operon an inducible operon • A repressor usually controls an inducible gene or operon by keeping it “turned off ” • The presence of an inducer inactivates the repressor, permitting the gene or operon to be transcribed • Inducible genes or operons usually code for enzymes that break down molecules (catabolic reactions) • This saves the energy cost of making enzymes when no substrates are available Repressible Genes • Repressible operons and genes are usually “turned on” – they are turned off only under certain conditions • Repressible genes usually code for enzymes that synthesize essential biological molecules from simpler materials (anabolic reactions) • The molecular signal for regulating these genes usually is the end product of the anabolic pathway The trp Operon • The tryptophan operon (trp operon) is an example of a repressible system • A repressor gene codes for a repressor protein which is synthesized in an inactive form (it can’t bind to the operator region of the trp operon) • When tryptophan binds to the repressor, the repressor is able to binds to the operator, which switches the operon off – transcription is blocked The trp Operon Repressor gene trp operon Promoter Operator trpE trpD trpC trpB trpA DNA mRNA RNA polymerase Transcription mRNA Translation Repressor protein (inactive) Enzymes of the tryptophan biosynthetic pathway Tryptophan (a) Low intracellular tryptophan levels. Repressor protein is unable to prevent transcription because it cannot bind to the operator. Fig. 14-4a, p. 312 trp operon Repressor gene DNA Promoter Operator trpE trpD trpC trpB trpA Active repressor – corepressor complex mRNA Inactive repressor protein Tryptophan (corepressor) (b) High intracellular tryptophan levels. The amino acid tryptophan binds to an allosteric site on the repressor protein, changing its conformation. The resulting active form of the repressor binds to the operator region, blocking transcription of the operon until tryptophan is again required by the cell. Fig. 14-4b, p. 312 Negative Regulators and Positive Regulators • The lac and trp operons are examples of negative control, a regulatory mechanism in which the DNA binding regulatory protein is a repressor that turns off transcription of the gene • Positive control is regulation by activator proteins that bind to DNA and thereby stimulate the transcription of a gene • The lac operon is controlled by both a negative regulator (the lactose repressor) and a positively acting activator protein Positive Control of the lac Operon • A DNA sequence near the promoter binds another regulatory protein, the catabolite activator protein (CAP) • When activated, CAP stimulates transcription of the lac operon and several other bacterial operons • In its active form, CAP is bound by an allosteric site to cyclic AMP (cAMP) • cAMP levels increase as cells become depleted of glucose Positive Control (cont.) • Activated CAP binds to the CAP binding site near the lac operon promoter • Binding strengthens the affinity of the promoter region for RNA polymerase – so the rate of transcriptional initiation accelerates in the presence of lactose • The lac operon is fully active only if lactose is available and intracellular glucose levels are low Positive Control of the lac Operon Promoter Repressor gene RNA CAP- polymerase – binding binding site site Operator lacZ lacY lacA DNA mRNA RNA polymerase binds poorly CAP (inactive) Allolactose Repressor protein (inactive) (a) Lactose high, glucose high, cAMP low. When glucose levels are high, cAMP is low. CAP is in an inactive form and cannot stimulate transcription. Transcription occurs at a low level or not at all. Fig. 14-5a, p. 313 Promoter Repressor gene RNA CAP- polymerase – binding binding site site Operator lacZ lacY lacA DNA mRNA RNA polymerase binds poorly CAP (inactive) Allolactose Repressor protein (inactive) (a) Lactose high, glucose high, cAMP low. When glucose levels are high, cAMP is low. CAP is in an inactive form and cannot stimulate Stepped Art transcription. Transcription occurs at a low level or not at all. Fig. 14-5a, p. 313 Promoter Repressor gene RNA polymerase – binding CAPsite binding site Operator DNA lacZ lacY lacA RNA polymerase Transcription binds efficiently mRNA mRNA CAP cAMP Allolactose Repressor protein (inactive) Translation Galactoside transacetylase Lactose permease β -galactosidase Enzymes for lactose metabolism (b) Lactose high, glucose low, cAMP high. When glucose concentrations are low, each CAP polypeptide has cAMP bound to its allosteric site. The active form of CAP binds to the DNA sequence, and transcription becomes activated. Fig. 14-5b, p. 313 Promoter Repressor gene RNA polymerase – binding CAPsite binding site Operator DNA lacZ lacY lacA RNA polymerase Transcription binds efficiently mRNA mRNA CAP cAMP Allolactose Repressor protein (inactive) Translation Galactoside transacetylase Lactose permease β -galactosidase Enzymes for lactose metabolism (b) Lactose high, glucose low, cAMP high. When glucose concentrations are low, each CAP polypeptide has cAMP bound to its allosteric site. The active form of CAP binds to the DNA sequence, and transcription becomes activated. Stepped Art Fig. 14-5b, p. 313 Binding of CAP to DNA Transcriptional Control in Bacteria Table 14-1, p. 314 Transcription of Constitutive Genes • Constitutive genes are continuously transcribed, but not all are transcribed at the same rate • Promoter elements control transcription rate • “Strong” promoters bind RNA polymerase more frequently and transcribe more mRNA molecules than those with “weak” promoters Posttranscriptional Regulation in Bacteria • Posttranscriptional controls are regulatory mechanisms that occur after transcription • Translational controls regulate the rate at which an mRNA molecule is translated • Posttranslational controls act as switches that activate or inactivate one or more existing enzymes • Example: feedback inhibition KEY CONCEPTS 14.2 • Gene regulation in bacteria occurs primarily at the level of transcription; regulation of transcription can be either positive or negative 14.3 GENE REGULATION IN EUKARYOTIC CELLS LEARNING OBJECTIVES: • Discuss the structure of a typical eukaryotic gene and the DNA elements involved in regulating that gene • Give examples of some of the ways eukaryotic DNA-binding proteins bind to DNA • Illustrate how a change in chromosome structure may affect the activity of a gene • Explain how a gene in a multicellular organism may produce different products in different types of cells • Identify some of the types of regulatory controls that operate in eukaryotes after mature mRNA is formed Gene Regulation in Eukaryotes • Eukaryotes have transcriptional, posttranscriptional, translational, and posttranslational gene controls that allow individual cells, tissues, and organs to function • Eukaryotic genes are not typically arranged in operon-like clusters – each eukaryotic gene has specific regulatory sequences that control transcription • Some genes are constitutive, others are inducible Inducible Genes • Some inducible genes respond to environmental threats such as heavy-metal ingestion, viral infection, and heat shock • Example: Some heat-shock proteins are molecular chaperones that help proteins fold into their proper shape • Some genes are inducible only during certain periods in the life of the organism (controlled by temporal regulation) • Some genes are under tissue-specific regulation Chromosome Organization • Chromosome organization affects expression of some genes • In multicellular eukaryotes, only a subset of genes in any specific type of cell is active – in many cases, inactivated genes are irreversibly dormant • Some inactive genes lie in highly compacted heterochromatin, such as the inactive X chromosomes in female mammals (Barr body) Chromosome Organization (cont.) • Active genes are associated with euchromatin, which is loosely packed and allows interaction with transcription factors and other regulatory proteins • Chromatin can be changed between heterochromatin and euchromatin by chemically modifying histones, the proteins that associate with DNA to form nucleosomes Regulation of Chromatin Gene Inactivation by DNA Methylation • Once a gene has been turned off by some other means, DNA methylation ensures it will remain inactive • Enzymes add methyl groups to certain cytosine nucleotides in DNA, which blocks transcription • DNA methylation can be maintained for multiple generations, as in genomic imprinting (parental imprinting), in which expression of certain genes is determined by whether the allele is inherited from the female or the male parent Genomic Imprinting and Epigenetics • Methylation provides a mechanism for epigenetic inheritance (changes in how a gene is expressed) • New phenotypic traits can arise from epigenetic changes despite the fact that the nucleotide sequence of the gene itself has not changed • Tumor suppressor genes that inhibit cell division (and cancer) are sometimes inactivated epigenetically, resulting in cancer Epigenetic Variation in the agouti Gene Fig. 14-9, p. 316 Increasing the Number of Copies of a Gene • A single gene may not provide enough copies of its mRNA to meet the cell’s needs • Genes required only by a small group of cells may be selectively replicated by gene amplification Drosophila chorion gene Gene amplification by repeated DNA replication of chorion gene region Chorion gene in ovarian cell Fig. 14-10, p. 317 Functional Promoter Elements • Transcription requires a base pair where transcription begins (transcription initiation site or start site) plus a sequence of bases to which RNA polymerase binds (promoter) • Certain promoter elements have regulatory functions and facilitate expression of the gene – mutations in these elements reduce the rate of transcription • Example: The TATA box, located about 25 to 30 base pairs upstream from the transcription initiation site Enhancers and Silencers • Enhancers and silencers regulate a gene on the same DNA molecule from very long distances • enhancers • DNA sequences that help form an active transcription initiation complex • silencers • DNA sequences that can decrease transcription Elements That Regulate Transcription • Eukaryotic regulatory elements include the promoter and various enhancers and silencers DNA Upstream double helix Silencer TATA box Enhancer Promoter Downstream Gene Fig. 14-11, p. 317 Transcription Factors • Many DNA-binding proteins that regulate transcription (transcription factors) have been identified in humans • Each eukaryotic transcription factor has a DNA-binding domain plus at least one other domain that is either an activator or a repressor of transcription for a given gene Transcription Factors (cont.) • Transcription in eukaryotes requires multiple regulatory proteins that are bound to different parts of the promoter • The general transcriptional machinery binds to the TATA box of the promoter, and is required for RNA polymerase to bind • An activator has at least two functional domains: • A DNA recognition site that binds to an enhancer • An activation site that contacts the target in the general transcriptional machinery Stimulation of Transcription by an Enhancer Enhancer Target proteins RNA polymerase TATA box DNA (a) Low rate of transcription. This gene is transcribed at a basic level when the general transcriptional machinery, including RNA polymerase, is bound to the promoter. Fig. 14-13a, p. 319 Enhancer DNA TATA box Activator (transcription factor) (b) High rate of transcription. A transcription factor that functions as an activator binds to an enhancer. The intervening DNA forms a loop, allowing the transcription factor to contact one or more target proteins in the general transcriptional machinery, thereby increasing the rate of transcription. This diagram is highly simplified, and many more target proteins than the two shown are involved. Fig. 14-13b, p. 319 Posttranscriptional Control • Eukaryotic mRNA molecules are capped, polyadenylated (addition of a poly-A tail), spliced, and then transported from the nucleus to the cytoplasm to initiate translation • Each of these events represent potential control points • Example: When the short poly-A tail of an mRNA is elongated, mRNA becomes activated and is translated Pre-mRNA Processing • Alternative splicing patterns allow the same gene to produce one type of protein in one tissue and a different type of protein in another tissue • Typically, such a gene includes at least one segment that can be either an intron or an exon Alternative Splicing Exon Potential splice sites Exon or intron Intron Exon pre-mRNA Alternative splicing Exon Exon Exon Functional mRNA in tissue A Exon Exon Functional mRNA in tissue B Fig. 14-14, p. 319 Stability of mRNA Molecules • Controlling the lifespan of an mRNA molecule regulates the number of protein molecules translated from it • In some cases, mRNA stability is under hormonal control Posttranslational Chemical Modifications • Another way to control gene expression is by regulating the activity of the gene product • In proteolytic processing, proteins are synthesized as inactive precursors, which are converted to an active form by removal of a portion of the polypeptide chain • Chemical modification (adding or removing functional groups) reversibly alters the activity of an enzyme - Enzymes that add phosphate groups are called kinases; those that remove them are phosphatases Posttranslational Chemical Modifications (cont.) • Posttranslational control of gene expression can also involve protein degradation • Proteins that are selectively targeted for degradation in a proteasome are covalently bonded to ubiquitin • Proteasomes are large macromolecular structures that recognize ubiquitin tags • Proteases (protein-degrading enzymes) associated with proteasomes hydrolyze peptide bonds Protein Degradation • As protein is degraded, ubiquitin molecules are released intact and reused Gene Regulation in Eukaryotic Cells Cytosol Nucleus Regulation of chromatin DNA must be unpacked; heterochroma- tin cannot be transcribed. Methylated DNA is inaccessible to transcription machinery; demethylated DNA can be transcribed. Regulation of transcription Selective transcription: promoter and enhancer elements in DNA interact with protein transcription factors to activate or repress transcription. Regulation of mRNA processing Control mechanisms, such as rate of intron/exon splicing, regulate mRNA processing. Alternative splicing of exons produces different proteins from same mRNA. Regulation of mRNA transport Controlling access to, or efficiency of, transport through nuclear pores regulates mRNA transport from nucleus to cytosol. Nuclear pore Regulation of translation Translational controls determine how often and how long specific mRNA is translated. Modifications to protein Chemical modifications, such as phosphorylation, affect activity of protein after it is produced. Degradation of mRNA Translational controls determine degree to which mRNA is protected from destruction; proteins that bind to mRNA in cytosol affect stability. Degradation of protein Selective degradation targets specific proteins for destruction by proteasomes. Fig. 14-7, p. 315 KEY CONCEPTS 14.3 • Gene regulation in eukaryotes occurs at the levels of transcription, posttranscription, translation, and posttranslation