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Chapter 23 The Regulation of Gene Expression Lectures by Kathleen Fitzpatrick Simon Fraser University © 2012 Pearson Education, Inc. The Regulation of Gene Expression • Regulation is an important aspect of almost every process in nature, especially gene expression • Selective gene expression allows cells to be efficient, synthesizing only what is needed for each cell type • The first understanding of gene regulation came from bacteria © 2012 Pearson Education, Inc. Bacterial Gene Regulation • Some genes are constitutive, expressed all the time • For most others, expression is regulated to meet the cell’s needs • Regulated genes encode enzymes needed for processes that are not constantly required © 2012 Pearson Education, Inc. Catabolic and Anabolic Pathways Are Regulated Through Induction and Repression, Respectively • Bacteria use different approaches for regulating enzyme synthesis depending on whether an enzyme is involved in a catabolic (degradative) or anabolic (synthetic) pathway • Enzymes that catalyze such pathways are often regulated coordinately © 2012 Pearson Education, Inc. Catabolic Pathways and Substrate Induction • Catabolic enzymes exist for the purpose of degrading specific substrates • In the catabolic pathway that degrades lactose, the central step in the pathway is catalyzed by -galactosidase • Before lactose can be hydrolyzed, it must be transported into the cell via a protein called galactosidase permease © 2012 Pearson Education, Inc. Substrate induction • -galactosidase is needed only when lactose is present • Therefore, it is turned on, induced, only in the presence of lactose, an example of substrate induction • Enzymes whose synthesis is regulated this way are called inducible enzymes © 2012 Pearson Education, Inc. Anabolic Pathways and End-Product Repression • For anabolic pathways, the amount of enzyme produced by a cell usually correlates inversely with the concentration of the end product of the pathway • E.g., as the concentration of tryptophan rises, it is efficient for the cell to reduce the production of the enzymes needed for tryptophan synthesis • This is end-product repression © 2012 Pearson Education, Inc. Repression and inhibition • Repression is a general term, referring to reduced expression of any regulated gene • Genetic repression has an effect on protein synthesis, not just protein activity • In feedback inhibition molecules of enzyme are still present but their catalytic activity is inhibited © 2012 Pearson Education, Inc. Effector Molecules • In both induction and repression of enzyme synthesis, control is triggered by small organic molecules, present in the cell or its surroundings • The small organic molecules are called effectors © 2012 Pearson Education, Inc. The Genes Involved in Lactose Catabolism Are Organized into an Inducible Operon • The cornerstone of the lactose operon model rests on the discovery of two types of genes • The first are the genes coding for enzymes used for lactose uptake and metabolism • The second is a regulatory gene whose product controls activity of the first group © 2012 Pearson Education, Inc. Genes for lactose metabolism • The lacZ gene encodes -galactosidase, which hydrolyzes lactose • The lacY gene encodes galactosidase permease, involved with uptake of lactose from outside the cell • The lacA gene encodes a transacetylase that adds an acetyl group to lactose as it enters the cell © 2012 Pearson Education, Inc. The operon • The three lac genes belong to a single regulatory unit, an operon • An operon is a group of genes with related function that are clustered together and are turned on and off simultaneously • This is less common in eukaryotes than in prokaryotes © 2012 Pearson Education, Inc. The lac Operon Is Negatively Regulated by the lac Repressor • In an operon, genes with related functions are clustered together so their transcription can be coordinately regulated • In order for inducers such as lactose to turn on the genes, an additional gene is needed, the lacI gene • In the absence of the lacI gene, cells always produce the lacZ, lacY, and lacA gene products, even when lactose is not present © 2012 Pearson Education, Inc. The lac operon • The lacI gene codes for a product that regulates expression of the lacZ, lacY, and lacA genes • A regulatory gene product that inhibits the expression of other genes is called a repressor protein • The lac operon consists of the three above genes preceded by a promoter (Plac) and a sequence called the operator (O) © 2012 Pearson Education, Inc. Transcription of the lac operon • Transcription of the operon begins at the promoter and proceeds through the operator and the lacZ, lacY, and lacA genes • The result is one transcript that codes for all three gene products • mRNAs that code for more than one polypeptide are called polycistronic mRNAs © 2012 Pearson Education, Inc. Figure 23-2 © 2012 Pearson Education, Inc. Control of the lac operon • The interaction between the operator site and the repressor protein is crucial to lac operon control • The lacI gene encodes the lac repressor, and is located just outside the operon • The repressor binds the operator, preventing RNA polymerase from transcribing the lac genes © 2012 Pearson Education, Inc. Figure 23-3A © 2012 Pearson Education, Inc. Regulation of the lac operon • Inducer molecules bind to the repressor, altering its conformation so that it cannot bind the operator any longer • Once the operator is no longer blocked by the repressor, the RNA polymerase can transcribe the lacZ, lacY, and lacA genes • The repressor is an allosteric protein, with two possible conformational states © 2012 Pearson Education, Inc. Figure 23-3B © 2012 Pearson Education, Inc. Allolactose is the inducer • The effector for the repressor is allolactose, an isomer of lactose produced after lactose enters the cell • The binding of allolactose to the repressor induces a conformational change that renders the repressor incapable of binding the operator • In this way lactose triggers the induction of the lac operon © 2012 Pearson Education, Inc. Studying the lac operon • To study the lac operon, a synthetic inducer, -galactoside isopropylthiogalactoside (IPTG) is used as the inducing molecule • The term inducer is used to describe any effector that turns on transcription of an inducible operon • The lac operon is an inducible operon because it is turned off unless induced © 2012 Pearson Education, Inc. Studies of Mutant Bacteria Revealed How the lac Operon Is Organized • Much of the early evidence for the operon model involved studies of mutant bacteria • Some mutations were located in the operon genes, which affect only one of the protein products • Others occur in the regulatory elements of the operon that affect all of the genes coordinately © 2012 Pearson Education, Inc. Table 23-1 © 2012 Pearson Education, Inc. Operon Gene Mutations • Mutations in the lacY or lacZ genes lead to production of defective enzymes even in the presence of inducer • The mutants are unable to use lactose as a carbon source • A defective gene or regulatory sequence is indicated by a superscript minus sign, e.g., Y © 2012 Pearson Education, Inc. Operator Mutations • Mutations in the operator can lead to constitutive expression • In these, the mutant cells continue to produce the lac enzymes whether inducer is present or not • Operator-constitutive mutants are represented as Oc © 2012 Pearson Education, Inc. Promoter Mutations • Promoter mutations can decrease the affinity of RNA polymerase for the promoter so that the rate of mRNA production decreases • P mutations decrease both the elevated rate of enzymes produced in presence of inducer, and the low, basal level of production without inducer © 2012 Pearson Education, Inc. Regulatory Gene Mutations • There are two types of mutations in the lacI gene • Some mutants fail to produce any of the lac enzymes whether or not inducer is present • These are called superrepressor mutants (Is) • The repressor remains bound to the operator whether or not inducer is present © 2012 Pearson Education, Inc. Regulatory gene mutations (continued) • The other class of lacI mutations produces a repressor protein that does not recognize the operator (or is not synthesized at all) • The lac operon in such I mutants cannot be turned off, so that the enzymes are synthesized continuously © 2012 Pearson Education, Inc. The Cis-Trans Test Using Partially Diploid Bacteria • A cis-trans test is used to differentiate between cis-acting mutations, which affect DNA sites, and trans-acting mutations, which affect proteins • Partially diploid bacteria can be constructed by inserting a second copy of the lac portion of the bacterial chromosome into the F-factor of F+ cells © 2012 Pearson Education, Inc. Table 23-2 © 2012 Pearson Education, Inc. The cis-trans test • If only one copy of the operon contains an I or Oc mutation, it is possible to determine if the mutation affects both copies of the operon • A partial diploid can be made with one copy of the operon containing a Z mutation and the other copy the Y mutation • In this way, the effect of regulatory mutations can be studied © 2012 Pearson Education, Inc. The cis-trans test • Mutations that affect only the genes to which they are physically connected are said to be cis-acting • Mutations that can affect both copies of the operon are said to be trans-acting • E.g., I+ allows for proper gene expression even in the presence of an I mutation; thus the repressor is called a trans-acting factor © 2012 Pearson Education, Inc. O mutations are cis-acting • In contrast to the I gene, O locus mutants act in cis • E.g., Oc affects only genes physically connected to it; thus the O site is a cis-acting element • Cis-specificity is characteristic of mutations that affect binding sites on DNA rather than protein products © 2012 Pearson Education, Inc. Catabolite Activator Protein (CAP) Positively Regulates the lac Operon • Glucose is the preferred carbon source for almost all cells • Catabolite repression is used by bacteria to ensure that glucose is metabolized preferentially when available • It is an example of positive transcriptional control (as opposed to negative control of lac operon regulation) © 2012 Pearson Education, Inc. The effector molecule is cAMP • In catabolite repression, the effector molecule is cyclic AMP (cAMP) • Glucose inhibits adenylyl cyclase; the more glucose is present, the less cAMP is made • Catabolite activator protein (CAP) is an activator protein that turns on transcription and can bind DNA of some operons when it is complexed with cAMP © 2012 Pearson Education, Inc. CAP regulation of transcription • The CAP-binding site is located upstream of the promoter in the lac operon • Similar sites are found in the inducible operons for metabolism of other sugars • The binding of activated (cAMP-bound) CAP greatly enhances binding of RNA polymerase to the promoter, promoting operon transcription when glucose is absent © 2012 Pearson Education, Inc. Figure 23-4, Steps 1, 2 © 2012 Pearson Education, Inc. Figure 23-4, Steps 3, 4 © 2012 Pearson Education, Inc. The lac Operon Is an Example of the Dual Control of Gene Expression • The lac operon is subject to both negative and positive control • Most tightly regulated genes in eukaryotes and prokaryotes are under such dual control • Factors whose binding of DNA inhibit transcription are negative regulators; those that enhance transcription are positive regulators © 2012 Pearson Education, Inc. The Structure of the lac Repressor/Operator Complex Confirms the Operon Model • In 1996, Lewis, Lu, and colleagues reported the structure of the lac repressor protein bound to the operator site • This confirmed genetic and biochemical studies • The repressor interacts with the operator as a tetramer © 2012 Pearson Education, Inc. The repressor/operator complex • The primary operator (O1) is 21 bp long • There are two additional sequences called O2, O3; when repressor binds O1 and O3 a loop is formed that prevents RNA polymerase from moving along the operon • When allolactose binds the repressor, it releases the DNA, which relaxes and allows transcription © 2012 Pearson Education, Inc. Figure 23-5 © 2012 Pearson Education, Inc. Figure 23-5A © 2012 Pearson Education, Inc. Figure 23-5B © 2012 Pearson Education, Inc. Figure 23-5C © 2012 Pearson Education, Inc. The Genes Involved in Tryptophan Synthesis Are Organized into a Repressible Operon • Operons coding for enzymes involved in catabolic pathways generally resemble the lac operon in being inducible • Operons that regulate enzymes involved in anabolic pathways are repressible operons • They are turned off allosterically, usually by an effector that is the end-product of the pathway © 2012 Pearson Education, Inc. The trp operon • The trp operon contains genes coding for enzymes needed in tryptophan biosynthesis and the accompanying regulatory DNA sequences • The effector molecule is the end-product of the pathway, tryptophan • Production of trp biosynthetic enzymes is repressed in the presence of trp © 2012 Pearson Education, Inc. The trp repressor • The regulatory gene for the trp operon is called trpR; it encodes a repressor protein that is active when bound to the effector • Tryptophan can be considered a corepressor, because it is required, along with the repressor protein, to shut off expression of the operon © 2012 Pearson Education, Inc. Figure 23-6A © 2012 Pearson Education, Inc. Figure 23-6B © 2012 Pearson Education, Inc. Sigma Factors Determine Which Sets of Genes Can Be Expressed • Bacterial cells employ different sigma (s) factors to control which genes will be transcribed • s factors recognize gene promoters; the most common one in E. coli is s70, though there are others used when environmental changes occur • Alternative s factors alter promoter recognition, allowing cells to adapt to the changed conditions © 2012 Pearson Education, Inc. Sigma factors • Different types of bacteria have different numbers of alternative sigma factors • Some are produced by bacteriophages, which allows the phage to take over the transcriptional machinery of the cell © 2012 Pearson Education, Inc. Attenuation Allows Transcription to Be Regulated After the Initiation Step • Bacteria employ additional regulatory mechanisms that operate after transcription is initiated • The trp operon of E. coli has an unusual leader sequence between the promoter and the first gene, trp E. • The leader sequence is transcribed even in the absence of trp © 2012 Pearson Education, Inc. Attenuation in the trp operon • A portion of the leader sequence is translated, forming a leader peptide 14 amino acids long • The leader sequence mRNA contains two adjacent codons for trp • The leader also has 4 regions (numbered 1–4) that can pair to form distinctive hairpin loops © 2012 Pearson Education, Inc. Figure 23-7 © 2012 Pearson Education, Inc. Attenuation in the trp operon (continued) • Regions 3 and 4 of the leader mRNA plus eight adjacent Us comprise the terminator • When trp levels are low, a ribsome that reaches the trp codons will pause, awaiting the tRNA trp • The stalled ribosome blocks region I, promoting a hairpin structure to form between regions 2 and 3 (the antiterminator hairpin) © 2012 Pearson Education, Inc. Attenuation in the trp operon (continued) • When region 3 is paired with region 2, it cannot form the termination structure with region 4, so transcription continues • However, if trp is plentiful, the ribosome does not pause and continues to the stop codon in region 2 • This permits interaction between regions 3 and 4, which terminates transcription © 2012 Pearson Education, Inc. Figure 23-8A © 2012 Pearson Education, Inc. Figure 23-8B © 2012 Pearson Education, Inc. Figure 23-8C © 2012 Pearson Education, Inc. Attenuation is fairly common • Although attenuation was once considered to be an unusual type of regulation, it is relatively common • This is especially true for operons involved in amino acid biosynthesis • In some operons, attenuation complements regulation via the operator; in others it is the only means of regulation © 2012 Pearson Education, Inc. Riboswitches Allow Transcription and Translation to Be Controlled by Small Molecule Interactions with RNA • Small molecules can regulate gene expression by binding to special sites in mRNA called riboswitches • Binding of the molecule to its riboswitch triggers changes in mRNA shape that affect transcription or translation • Riboswitches are typically found in the untranslated leader region of mRNAs of bacterial operons © 2012 Pearson Education, Inc. One of the first riboswitches to be discovered • One of the first riboswitches identified is RNA from the riboflavin (rib) operon of the bacterium B. subtilis • The RNA of the rib operon possesses a leader sequence that can fold into a hairpin loop that terminates transcription • The coenzymes FMN and FAD are end-products of the operon; binding of FMN to the leader sequences promotes formation of a termination hairpin © 2012 Pearson Education, Inc. Figure 23-9A © 2012 Pearson Education, Inc. Riboswitches can control translation • The binding of small molecules to riboswitches can also control translation • The genes involved in synthesizing FMN and FAD in E. coli are not clustered in an operon but some are controlled by riboswitches • Binding of RMN to its riboswitch promotes formation of a hairpin that includes sequences required for binding the mRNA to ribosomes © 2012 Pearson Education, Inc. Figure 23-9B © 2012 Pearson Education, Inc. Eukaryotic Gene Regulation: Genomic Control • In some fundamental features, there are numerous similarities between bacteria and eukaryotes • However, eukaryotes require a diversity of genetic control mechanisms, some of which are quite different from those of bacteria © 2012 Pearson Education, Inc. Multicellular Eukaryotes Are Composed of Numerous Specialized Cell Types • In the case of multicellular eukaryotes, a single organism consists of a mixture of specialized or differentiated cell types • These are distinguished from each other based on difference in appearance and protein products • Such differences indicate that differential gene expression plays a central role in creating differentiated cells © 2012 Pearson Education, Inc. Differentiation • Differentiated cells are produced from groups of immature, nonspecialized cells • The process is called cell differentiation • The classic example occurs in embryos, in which cells of the early embryo produce all the cell types that make up the organism © 2012 Pearson Education, Inc. Eukaryotic Gene Expression Is Regulated at Five Main Levels • The overall pattern of gene expression results from controls exerted at several different levels • Control is exerted at five levels: the genome (1), transcription (2), RNA processing and export (3), translation (4), and posttranslational events (5) • The last three categories represent levels of posttranscriptional control © 2012 Pearson Education, Inc. Figure 23-10, Parts 1–3 © 2012 Pearson Education, Inc. Figure 23-10, Parts 4, 5 © 2012 Pearson Education, Inc. As a General Rule, the Cells of a Multicellular Organism All Contain the Same Set of Genes • The first level of control is exerted at the level of the overall genome • Though each specialized cell type uses only a fraction of the genes in the genome, almost all cells retain the same complete set of genes • In 1958, this was demonstrated by Steward, who grew entire carrot plants from single root cells © 2012 Pearson Education, Inc. More evidence from the African clawed frog (Xenopus laevis) • In 1964, Gurdon and colleagues transplanted nuclei from differentiated tadpole cells into unfertilized eggs deprived of their own nuclei • The frequency of success was low, but some of the eggs developed into swimming tadpoles • These tadpoles, created by nuclear transplantation, were clones of the individual from which the original nucleus was taken © 2012 Pearson Education, Inc. Cloning mammals • In 1997 Wilmut and colleagues cloned a sheep, Dolly, the first mammal cloned from a cell that came from an adult • Similar techniques have been used to clone other mammals • Nuclei that contain a complete set of genes, and can direct formation of a new organism, are said to be totipotent © 2012 Pearson Education, Inc. Figure 23A-1A, Steps 1, 2 © 2012 Pearson Education, Inc. Figure 23A-1A, Steps 3, 4 © 2012 Pearson Education, Inc. Figure 23A-1A, Steps 5, 6 © 2012 Pearson Education, Inc. Figure 23A-1B © 2012 Pearson Education, Inc. Gene Amplification and Deletion Can Alter the Genome • A few types of gene regulation create exceptions to the rule that the genome tends to be identical in all the cells of an adult eukaryote • Gene amplification is used to make multiple copies of the same gene • This is an example of genomic control, a regulatory change in the makeup or organization of a genome © 2012 Pearson Education, Inc. Amplification of rRNA genes in Xenopus laevis • The haploid genome of Xenopus has about 500 copies of genes for 5.8S, 18S, and 28S rRNA • However, the genes are selectively replicated about 4000-fold during oogenesis so that the mature oocyte contains about 2 million copies • This amplification is needed to produce enough ribosomes during oogenesis, enough to sustain the embryo through its early development © 2012 Pearson Education, Inc. Gene deletion also occurs • Some cells delete genes whose products are not required • Extreme gene deletion (DNA diminution) occurs in mammalian red blood cells • These discard their nuclei once sufficient hemoglobin RNA is synthesized © 2012 Pearson Education, Inc. DNA Rearrangements Can Alter the Genome • In a few cases, gene regulation is based on the movements of DNA segments from one location to another within the genome • This is called DNA rearrangement © 2012 Pearson Education, Inc. Yeast Mating-Type Rearrangements • In yeast, Saccharomyces cerevisiae mating occurs when haploid cells of different mating types (a and ) fuse to form a diploid cell • Cells have alleles for both mating types, but the actual mating type depends on which of the alleles is present at a special site in the genome called the MAT locus • Cells switch mating type by moving the alternate allele into the MAT locus © 2012 Pearson Education, Inc. The cassette mechanism • The process of DNA rearrangement involved in mating type switching is called the cassette mechanism • The MAT locus containing the a or allele is on chromosome 3, between extra copies of the two alleles • The extra copies are called HML and HMRa © 2012 Pearson Education, Inc. Mechanism of switching • To switch mating type, the HO endonuclease creates a break in the chromosome at the MAT locus • Endonucleases degrade the cut DNA • HML or HMRa DNA is used as a template to repair the gap in the DNA, which results in a switch of the mating type allele found at the MAT locus © 2012 Pearson Education, Inc. No expression from the HML and HMR genes • Though the yeast cells contain genes for both mating type proteins, only the one from the MAT locus is expressed • A group of regulatory proteins, the silent information regulator (SIR) genes, prevent expression at HML and HMR © 2012 Pearson Education, Inc. Figure 23-11 © 2012 Pearson Education, Inc. Antibody Gene Rearrangements • Lymphocytes of the vertebrate immune system undergo DNA rearrangement to produce antibodies • Antibodies are composed of heavy chains and light chains • Millions of different kinds of antibodies can be made by the vertebrate immune system © 2012 Pearson Education, Inc. Antibody production • Lymphocytes use a relatively small number of DNA segments and rearrange them in various combinations to produce millions of unique antibody genes • Four kinds of DNA segments are involved: V, J, D, and C segments • The C segment encodes constant regions of the heavy or light chains, the others are variable regions © 2012 Pearson Education, Inc. Antibody production (continued) • Heavy chains are constructed from about 200 kinds of V segments, more than 20 kinds of D segments, and at least 6 kinds of J segments • These are rearranged during lymphocyte development, when one of each type of segment is brought together at random, to generate at least 24,000 different kinds of heavy chain variable regions © 2012 Pearson Education, Inc. Antibody production (continued) • Light chains are similarly variable, though they do not use D segments • Further variation is produced as any light chain is assembled with any type of heavy chain to create millions of different combinations © 2012 Pearson Education, Inc. Figure 23-12 © 2012 Pearson Education, Inc. Chromosome Puffs Provide Visual Evidence That Chromatin Decondensation Is Involved in Genomic Control • In eukaryotes, some degree of chromatin decondensation is necessary for gene expression • Evidence of decondensation came from microscopic visualization of certain insect chromosomes • In the fruit fly, tissues such as the salivary glands contain large polytene chromosomes © 2012 Pearson Education, Inc. Polytene chromosomes • Polytene chromosomes form by successive replication without subsequent division • The newly formed chromatids line up in parallel to form the multistranded polytene chromosomes • These large chromosomes have a characteristic banding pattern and are easily visualized microscopically © 2012 Pearson Education, Inc. Chromosome puffs • Transcriptional activation of genes in a particular chromosome band causes the compacted chromatin to uncoil and expand • A visible chromosome puff is formed • Puffs are not the only sites of transcription, but the extent of decondensation at puff sites correlates well with the level of transcription © 2012 Pearson Education, Inc. Chromosome puffs and ecdysone • As the fruit fly (Drosophila) larvae progress through various stages, the pattern of chromosome puffing changes • The puffs respond to the steroid hormone, ecdysone • It binds to and activates a regulatory protein that stimulates expression of certain genes at certain times © 2012 Pearson Education, Inc. Figure 23-13 © 2012 Pearson Education, Inc. DNAse I Sensitivity Provides Further Evidence for the Role of Chromatin Decondensation in Genomic Control • DNAse I is an endonuclease that preferentially degrades transcriptionally active DNA in chromatin • The increased sensitivity of such regions to digestion suggests that their DNA is uncoiled • E.g., the globin gene is sensitive to low levels of DNAse I in chicken red blood cells (erythrocytes), where it is expressed, but not in other cells © 2012 Pearson Education, Inc. Figure 23-14 © 2012 Pearson Education, Inc. DNAse I hypersensitivity • When nuclei are treated with very low levels of DNAse I, specific regions that are very susceptible to digestion can be detected • These DNAse I hypersensitive sites tend to occur up to a few hundred bases upstream from the transcription start sites of active genes • These sites correspond to regions where DNA is nucleosome free © 2012 Pearson Education, Inc. Figure 23-15 © 2012 Pearson Education, Inc. DNA Methylation Is Associated with Inactive Regions of the Genome • DNA methylation, the addition of methyl groups to certain cytosine bases in DNA, can reduce gene activity • Methylation of promoter regions can block access of proteins needed for transcription, or serve as binding sites for proteins that condense chromatin • The net effect is gene silencing © 2012 Pearson Education, Inc. DNA methylation • DNA methylations occur on cytosines in 5-CG-3 sequences base-paired to 3-GC-5 sequences that are already methylated • This allows methylation patterns on DNA to be inherited • This can create epigenetic changes, stable alterations in gene expression, transmitted from one cellular generation to the next, with no change in DNA sequence © 2012 Pearson Education, Inc. X-inactivation • Female mammals have two X chromosomes, whereas males have only one • One of the X chromosomes in females is randomly inactivated during early embryonic development • In X-inactivation, the DNA of one X is extensively methylated and the chromatin condenses into a tight mass of heterochromatin, inaccessible to transcription factors © 2012 Pearson Education, Inc. X-inactivation • The inactivated X chromosome is visible as a dark spot called a Barr body • Once a certain X is inactivated in a cell, all the daughter cells produced by the original have the same X inactivated • The result of X-inactivation is that females and males each have one active X chromosome per adult cell © 2012 Pearson Education, Inc. Methylation and restriction enzymes • Evidence of connection of DNA methylation and gene inhibition comes from studies using the restriction enzymes HpaII and MspI • Both enzymes cleave the recognition site -CCGGbut HpaII cuts only if the central C is unmethylated • Comparing fragments generated by digesting with both enzymes shows that DNA sites are methylated in a tissue-specific fashion © 2012 Pearson Education, Inc. Figure 23-16 © 2012 Pearson Education, Inc. Figure 23-16A © 2012 Pearson Education, Inc. Figure 23-16B © 2012 Pearson Education, Inc. Imprinting and methylation • Genomic imprinting causes certain genes to be expressed differently depending on the parent from which they are inherited • This differing behavior results from differential methylation • Some genes are maternally imprinted (methylated) so only the paternal copy is active; and vice versa for paternally imprinted genes © 2012 Pearson Education, Inc. Imprinting syndromes • Prader-Willi syndrome is characterized by deletion of paternal genes (the maternal copies of which are methylated) from a particular region of chromosome 15 • Angelman syndrome results when a gene in this region is deleted from the maternal chromosome; the paternal copy of the gene is inactive © 2012 Pearson Education, Inc. Changes in Histones and Chromatin Remodeling Proteins Can Alter Genome Activity • Histone proteins affect packaging of DNA; their protruding tails can be tagged by addition of phosphate, methyl, or acetyl group • Combinations of these tags create a histone code, a set of signals for modifying chromatin structure and gene activity • Methylation of lysine can serve as a signal for activation or repression of gene activity © 2012 Pearson Education, Inc. Histone methylation and acetylation • Methylation of lysine 4 of histone H3 is a mark of active genes, whereas methylation of lysine 9 and 27 is associated with gene silencing • Acetylation is accomplished by the enzyme histone acetyl transferase (HAT) and generally promotes chromatin decondensation • Histone deacetylase (HDAC) removes acetyl groups from histones © 2012 Pearson Education, Inc. Figure 23-17A © 2012 Pearson Education, Inc. Gene activation and repression • Repressor proteins which cause transcription to occur less frequently, recruit HDAC complexes to deacetylate histones • Activator proteins recruit HAT complexes • DNAse I digestion releases acetylated histones H3 and H4, suggesting that they are preferentially associated with active genes © 2012 Pearson Education, Inc. Histone H1 and HMG proteins • Studies show that transcriptionally active chromatin often lacks histone H1 • Histone H1 is required for folding chromatin into 30 nm fibers, suggesting that its absence helps maintain active chromatin in the 10 nm form • Active chromatin also has a large content of HMG (high-mobility group) proteins, non-histone proteins that bind selectively to active genes © 2012 Pearson Education, Inc. Chromatin remodeling • In addition to modifying histones, other proteins alter the position of nucleosomes along the DNA • These chromatin remodeling proteins use the energy of ATP hydrolysis to slide nucleosomes or cause their ejection from a region of chromatin • One important class of these proteins is the SWI/SNF family © 2012 Pearson Education, Inc. Figure 23-17B © 2012 Pearson Education, Inc. Eukaryotic Gene Regulation: Transcriptional Control • Transcriptional control is the second main level for controlling gene expression © 2012 Pearson Education, Inc. Different Sets of Genes Are Transcribed in Different Cell Types • Different proteins produced by two cell types in the same individual result from differential gene transcription • Nuclear run-on transcription experiments provide a shapshot of the transcription in a nucleus at a given moment • These experiments demonstrate that transcribed RNAs in different cell types are different © 2012 Pearson Education, Inc. Figure 23-18 © 2012 Pearson Education, Inc. DNA Microarrays Allow the Expression of Thousands of Genes to Be Monitored Simultaneously • Northern blotting is used to determine whether a particular gene is active in a particular cell type or tissue • An RNA sample is run on a gel to separate the molecules by size; the gel is blotted onto a special paper, which is then exposed to a labeled DNA probe for the sequence of interest © 2012 Pearson Education, Inc. Microarrays • A DNA micorarray is used to monitor expression of hundreds or thousands of genes simultaneously • A microarray is a thin chip spotted with thousands of DNA fragments corresponding to genes of interest • RNAs are isolated from a cell population and copied into single-stranded cDNA molecules © 2012 Pearson Education, Inc. Microarrays (continued) • The cDNAs are labeled with a fluorescent dye • The microarrays are exposed to the labeled DNAs, which will bind their complementary DNA • This approach can be used to compare the patterns of gene expression in different tissues; a green dye labels cDNAs from one cell type and a red dye the cDNAs from the other cell type © 2012 Pearson Education, Inc. Microarrays (continued) • The mixture of cDNAs is placed on the microarray, and binds to genes expressed in each cell type • Green spots indicate genes expressed only by one cell type; red spots indicate genes expressed only in the other cell type • Genes that are expressed with both types will be yellow (the color resulting from a mixture of the red and green labels) © 2012 Pearson Education, Inc. Figure 23-19 © 2012 Pearson Education, Inc. Proximal Control Elements Lie Close to the Promoter • The specificity of transcription is determined by proteins called transcription factors • General transcription factors are essential for transcription of all the genes transcribed by a given type of RNA polymerase • General transcription factors assemble with RNA polymerase at the core promoter, very close to the transcription startpoint © 2012 Pearson Education, Inc. General transcription factors and basal transcription • General transcription factors usually initiate transcription at low, basal rates • Most protein coding genes have short DNA sequences farther upstream to which additional transcription factors bind, improving the efficiency of the core promoter • Proximal control elements are sequences about 100–200 bases upstream of the core promoter © 2012 Pearson Education, Inc. Proximal control elements • The number, exact location and identities of proximal control elements varies with each gene, but three types are common –The CAAT box –The GC box –The octamer • Transcription factors that bind one of these are called regulatory transcription factors © 2012 Pearson Education, Inc. Figure 23-20 © 2012 Pearson Education, Inc. Enhancers and Silencers Are Located at Variable Distances from the Promoter • Distal control elements, located upstream or downstream from the genes they regulate, often lie far away from the promoter • Enhancers stimulate gene transcription, and silencers inhibit transcription • The position of these elements relative to the promoter can vary greatly © 2012 Pearson Education, Inc. Enhancers • A typical enhancer contains several different control elements within it, each a short DNA sequence that is a binding site for a different regulatory factor • Some of these sequences may be identical to proximal control elements • The regulatory factors that bind enhancers are called activators © 2012 Pearson Education, Inc. Studying enhancer function • To investigate the properties of enhancers, researchers use recombinant DNA techniques to alter enhancer location and orientation relative to the regulated gene • Enhancers can function at variable distances from the startpoint if the promoter is present • When either the promoter or enhancer is missing, transcription ceases © 2012 Pearson Education, Inc. Figure 23-21 © 2012 Pearson Education, Inc. Silencers and repressors • Silencers share many features of enhancers, but inhibit transcription rather than activating it • Regulatory transcription factors that bind to silencers are called repressors • Proteins produced by the SIR gene are examples of repressors © 2012 Pearson Education, Inc. Insulators • Silencers and enhancers can function far from the genes they regulate • This creates a complication if genes with different expression patterns reside near each other • DNA sequences called insulators are sometimes employed to prevent an enhancer or silencer from affecting the wrong genes © 2012 Pearson Education, Inc. Coactivators Mediate the Interaction Between Regulatory Transcription Factors and the RNA Polymerase Complex • Coactivators include chromatin remodeling proteins, and enzymes that modify histones • A multiprotein complex called Mediator acts as a bridge that binds to activator proteins associated with enhancers and to RNA polymerase • It links enhancers to components involved in transcription initiation © 2012 Pearson Education, Inc. Triggering gene activation • Activator proteins bind to their DNA control elements in the enhancer, forming a complex called the enhanceosome • One or more of the activator proteins cause the DNA to form a loop that brings the enhancer close to the core promoter (1) © 2012 Pearson Education, Inc. Figure 23-22, Steps 1, 2 © 2012 Pearson Education, Inc. Triggering gene activation (continued) • The activators interact with proteins such as chromatin remodeling proteins and HAT, that alter chromatin structure at the promoter (2) • Finally the activators bind to Mediator, which facilitates correct positioning of RNA polymerase and general transcription factors, allowing transcription to begin (3) © 2012 Pearson Education, Inc. Figure 23-22, Step 3 © 2012 Pearson Education, Inc. Multiple DNA Control Elements and Transcription Factors Act in Combination • The combinatorial model for gene regulation proposes that a small number of control elements and transcription factors act in combinations to establish patterns of gene expression in different cell types • Transcription of genes encoding tissue-specific proteins requires transcription factors or combinations of them that are unique to individual cell types © 2012 Pearson Education, Inc. Figure 23-23 © 2012 Pearson Education, Inc. Figure 23-23A © 2012 Pearson Education, Inc. Figure 23-23B © 2012 Pearson Education, Inc. Several Common Structural Motifs Allow Regulatory Transcription Factors to Bind to DNA and Activate Transcription • Regulatory transcription factors possess the ability to bind to a specific DNA sequence and the ability to regulate transcription • These activities reside in separate protein domains: the DNA-binding domain, and the transcription regulation domain (activation domain) • Domain swap experiments demonstrate the independence of the two domains © 2012 Pearson Education, Inc. Figure 23B-1 © 2012 Pearson Education, Inc. Categories of DNA-Binding Domains • Most regulatory transcription factors can be placed into one of a small number of categories based on the motif (secondary structure pattern) that makes up the DNA binding domain © 2012 Pearson Education, Inc. Helix-Turn-Helix Motif • The helix-turn-helix motif is one of the most common DNA-binding motifs in both eukaryotic and prokaryotic regulatory transcription factors • It consists of two -helices separated by a bend in the polypeptide chain • One -helix, the recognition helix, binds the DNA, and the second helix stabilizes the overall configuration © 2012 Pearson Education, Inc. Figure 23-24A © 2012 Pearson Education, Inc. Zinc Finger Motif • The zinc finger motif consists of an helix and a two-segment sheet held in place by the interaction of cysteine and histidine residues with a zinc ion • Zinc fingers protrude from the protein surface and act as points of contact between the protein and the DNA © 2012 Pearson Education, Inc. Figure 23-24B © 2012 Pearson Education, Inc. Leucine Zipper Motif • The leucine zipper motif is formed by the interaction between two polypeptide chains, each with regularly spaced leucine residues • The leucine interact with one another and interlock, causing the two helices to wrap around each other © 2012 Pearson Education, Inc. Figure 23-24C © 2012 Pearson Education, Inc. Helix-Loop-Helix Motif • Helix-loop-helix motifs contain a short helix connected by a loop to another longer helix • They contain hydrophobic regions that usually connect two polypeptides, either similar or different © 2012 Pearson Education, Inc. Figure 23-24D © 2012 Pearson Education, Inc. DNA Response Elements Coordinate the Expression of Nonadjacent Genes • Eukaryotic cells often need to activate a group of related genes at the same time • Eukaryotes use DNA control sequences called response elements to turn transcription on and off in response to a particular signal © 2012 Pearson Education, Inc. Steroid Hormone Receptors Act as Transcription Factors That Bind to Hormone Response Elements • Steroid receptors and the related retinoid receptors belong to the zinc finger class of transcription factors • They typically consist of three domains: one recognizes and binds to a specific response element in DNA, a second binds a particular hormone, and the third activates transcription © 2012 Pearson Education, Inc. Hormone response elements • Steroid receptors bind to DNA sequences called hormone response elements • All genes activated by a particular steroid hormone are associated with the same type of response element • E.g. genes activated by estrogen have an estrogen response element; genes activated by glucocorticoids are next to glucocorticoid response elements © 2012 Pearson Education, Inc. Figure 23-25A © 2012 Pearson Education, Inc. Cortisol function • The GR cannot enter the nucleus as long as it is bound to the Hsp proteins • Binding of cortisol to the GR triggers release of Hsp and the GR with the bound cortisol enters the nucleus to bind glucocorticoid response elements • Binding of one GR to the response element facilitates binding of a second © 2012 Pearson Education, Inc. Cortisol function • The GR dimer activates transcription of the adjacent gene by recruiting coactivators that promote transcription • The glucocorticoid receptor contains an inverted repeat, which facilitates binding of two GRs • Cortisol activates all genes with glucocorticoid response elements simultaneously © 2012 Pearson Education, Inc. CREBs and STATs Are Examples of Transcription Factors Activated by Phosphorylation • Another approach for controlling transcription factor activity is based on protein phosphorylation • cAMP in eukaryotes functions by stimulating activity of protein kinase A, which in turn phosphorylates a variety of proteins • One target is CREB, which normally binds DNA sequences called cAMP response elements © 2012 Pearson Education, Inc. CREB and CBP • Once phosphorylated, CREB binds a coactivator called CBP (CREB-binding protein) • CBP catalyzes histone acetylation, loosening the packing of nucleosomes, and facilitates assembly of the transcription machinery at nearby promoters © 2012 Pearson Education, Inc. Figure 23-26 © 2012 Pearson Education, Inc. STATs • Another phosphorylation target is a family of transcription factors called STATs • Among the signaling molecules that activate STATs are interferons, glycoproteins secreted by animal cells in response to viral infections • Binding of interferon to its cell surface receptor causes cytosolic activation of the Janus kinase (Jak) © 2012 Pearson Education, Inc. STATs • Activated Jak phosphorylates STAT molecules in the cytoplasm • Phosphorylation causes STATs to dimerize and enter the nucleus where they bind target DNA response elements • The Jak-STAT signaling pathway shows considerable specificity © 2012 Pearson Education, Inc. Homeotic Genes Code for Transcription Factors That Regulate Embryonic Development • Homeotic genes are an unusual class of genes that when mutated cause transformation of one body structure into another – E.g., the bithorax gene complex, and the antennapedia gene complex • These genes each contain a similar 180-bp sequence near the 3 end, called the homeobox, which codes for the homeodomain © 2012 Pearson Education, Inc. Figure 23-27 © 2012 Pearson Education, Inc. Figure 23-27A © 2012 Pearson Education, Inc. Figure 23-27B © 2012 Pearson Education, Inc. Figure 23-27C © 2012 Pearson Education, Inc. Figure 23-28A © 2012 Pearson Education, Inc. Homeotic genes • The homeotic genes code for a family of regulatory transcription factors that activate or inhibit transcription of developmentally important genes • Each homeotic transcription factor can influence expression of dozens or hundreds of genes in the growing embryo © 2012 Pearson Education, Inc. Figure 23-28B © 2012 Pearson Education, Inc. Homeotic genes (continued) • The result of the coordinated gene regulation by homeotic genes is the establishment of fundamental body characteristics such as limb shape and location • Genes similar to these are found in a variety of organisms • The organization is also quite similar from one organism to the next © 2012 Pearson Education, Inc. Homeotic genes (continued) • In flies and vertebrates the 3 to 5 location of these genes corresponds to the anterior to posterior position along the body axis • The genes control identity of structures along the anterior-posterior axis • E.g., mutations in the HoxD13 locus result in synpolydactyly, fusions and duplications of fingers © 2012 Pearson Education, Inc. Figure 23-29 © 2012 Pearson Education, Inc. Eukaryotic Gene Regulation: Posttranscriptional Control • After transcription has taken place, the flow of genetic information involves a complex series of posttranscriptional events • Posttranscriptional regulation is especially useful in rapidly fine-tuning patterns of gene expression © 2012 Pearson Education, Inc. Control of RNA Processing and Nuclear Export Follows Transcription • Posttranscriptional control provides many opportunities for regulation of gene expression • RNA splicing is an especially important control site—its regulation allows cells to create a variety of different mRNAs from the same pre-mRNA • Alternative splicing permits some splice sites to be skipped and others activated © 2012 Pearson Education, Inc. Alternative splicing • Regulatory proteins and small nucleolar RNAs (snoRNAs) bind to splicing enhancer or splicing silencer sequences in pre-mRNA • E.g., the mRNA coding for the immunoglobulin M (IgM), which has two alternative poly(A) sites • Alternative splicing leads to the formation of mRNAs that code for either a secreted or membrane-bound version of the protein © 2012 Pearson Education, Inc. Figure 23-30 © 2012 Pearson Education, Inc. Nuclear export • Export of nuclei to the cytoplasm can be controlled—RNAs with defects in capping or splicing are not readily exported from the nucleus • Even with normal mRNAs certain molecules are retained until their export is triggered by a stimulus © 2012 Pearson Education, Inc. Translation Rates Can Be Controlled by Initiation Factors and Translational Repressors • Once mRNAs reach the cytoplasm, several translational control mechanisms regulate their rate of translation • Some mechanisms work by altering ribosomes or protein synthesis factors • Others work by regulating the activity/stability of the mRNA itself © 2012 Pearson Education, Inc. A well-studied example of translational control • Globin polypeptides are the main product of translation (>90%) in erythrocytes; their synthesis depends on the availability of heme • A protein kinase calledheme-controlled inhibitor (HCI) is inactive in the presence of heme • In the absence of heme HCI phosphorylates and inhibits eIF2, a protein required for initiating translation © 2012 Pearson Education, Inc. eIF4F is also regulated by phosphorylation • eIF4F is a multiprotein complex that binds the 5 mRNA cap and is regulated by phosphorylation • Adenovirus inhibits protein synthesis in infected cells by blocking phosphorylation of eIF4F that is normally required for its activation • Other viruses produce proteases that cleave eIF4F and inactivate it; viral mRNAs can still be translated by one of the eIF4F fragments © 2012 Pearson Education, Inc. A more specific type of translational control • Ferritin is an iron-storage protein whose synthesis is selectively stimulated in the presence of iron • The 5 untranslated leader sequence contains a 28 nucleotide segment, the iron response element (IRE) • This element forms a hairpin loop required for stimulation of production of ferritin © 2012 Pearson Education, Inc. Iron regulation of ferritin synthesis • When iron concentration is low, a protein called IREbinding protein binds to the IRE sequence in the ferritin mRNA, which prevents formation of an initiation complex • When more iron is present the protein binds iron and undergoes a conformational change and releases the IRE, allowing translation • The IRE-binding protein is an example of a translational repressor © 2012 Pearson Education, Inc. Figure 23-31A © 2012 Pearson Education, Inc. Figure 23-31B © 2012 Pearson Education, Inc. Translation Can Also Be Controlled by Regulation of mRNA Degradation • Translation rates are subject to control by alterations in mRNA stability • Degradation rates of mRNA can be measured by pulse-chase experiments © 2012 Pearson Education, Inc. Pulse-chase experiments • Cells are incubated in the presence of a radioactive compound that is incorporated into mRNA, then placed in a nonradioactive medium • The fate of radioactivity in the mRNA can be measured • The mRNA half-life is the time required for 50% of the initial radioactive RNA to be degraded © 2012 Pearson Education, Inc. mRNA stability • Eukaryotic mRNA stability varies widely • mRNAs with longer poly(A) tails are more stable than those with shorter tails • Short-lived mRNAs have AU-rich elements in the 3 untranslated region; these trigger removal of the poly(A) tail © 2012 Pearson Education, Inc. Role of iron • The transferrin receptor mediates iron uptake across the plasma membrane • When iron levels are low, the IRE-binding protein binds an IRE in the 3 UTR of the transferrin receptor mRNA • They protect it from degradation • High iron levels cause the IRE-binding protein to release the IRE, allowing degradation of the mRNA © 2012 Pearson Education, Inc. Figure 23-32A © 2012 Pearson Education, Inc. Figure 23-32B © 2012 Pearson Education, Inc. Two mechanisms for mRNA degradation • In the 3 to 5 pathway shortening of the poly(A) tail, degradation of the RNA chain is accomplished by the cytoplasmic exosome, a complex of several exonucleases, with the 5 cap degraded last • The 5 to 3 pathway involves shortening the tail at the 3 end, followed by removal of the cap and then degradation of the mRNA by mRNA processing bodies (P bodies) © 2012 Pearson Education, Inc. RNA Interference Utilizes Small RNAs to Silence the Expression of Genes Containing Complementary Base Sequences • mRNAs can be controlled by a class of small RNA molecules that inhibit their expression via complementarity • RNAi (RNA interference) is based on the ability of small RNAs to trigger mRNA degradation © 2012 Pearson Education, Inc. RNAi • Double-stranded RNA (from certain types of viruses or introduced artificially) knocks down the expression of specific genes • First, a cytoplasmic ribonuclease called Dicer cleaves the double-stranded RNA into short fragments about 21–22 bp long • The resulting fragments are called siRNAs (small interfering or silencing RNAs) © 2012 Pearson Education, Inc. RNAi • The siRNAs combine with a group of proteins to form an inhibitor of gene expression called RISC (RNA-induced silencing complex), in this case called the siRISC • One of the strands is degraded, and the remaining one binds the siRISC to a target mRNA by complementary base pairing • Slicer cleaves the target mRNA • In some cases siRISC may enter the nucleus and silence the gene by DNA methylation © 2012 Pearson Education, Inc. Figure 23-33 © 2012 Pearson Education, Inc. MicroRNAs Produced by Normal Cellular Genes Silence the Translation of Developmentally Important Messenger RNAs • MicroRNAs (miRNAs) are produced by genes found in almost all eukaryotes • These bind to and regulate expression of genes that are separate from the genes that produce the miRNAs • miRNAs are initially transcribed into longer molecules called primary microRNAs (pri-mRNAs), which fold into hairpin loops © 2012 Pearson Education, Inc. miRNAs • Looped pri-miRNAs are converted into mature miRNAs • A nuclear enzyme called Drosha cleaves the primiRNAs into smaller hairpins called precursor miRNAs, pre-miRNAs • The pre-miRNAs are exported to the cytoplasm, where Dicer cleaves them to form an miRNA © 2012 Pearson Education, Inc. miRNAs • The miRNA forms an miRISC, which inhibits expression of mRNAs containing sequences complementary to the miRNA • mRNAs with fully complementary sequences are degraded by miRISC • mRNAs with partially complementary sequences are translationally inhibited © 2012 Pearson Education, Inc. Figure 23-34 © 2012 Pearson Education, Inc. miRNA roles during development • MiRNAs play important roles in embryonic development • E.g. deleting an miRNA called miR-1-2 causes heart defects leading to embryonic lethality in mice • At least a dozen of the mRNAs targeted for degradation by miR-1-2 have been identified © 2012 Pearson Education, Inc. Posttranslational Control Involves Modification of Protein Structure, Function, and Degradation • The posttranslational control mechanisms include – – – – Structural alterations that influence protein function Guiding of protein folding Targeting to specific locations Interaction with regulatory molecules © 2012 Pearson Education, Inc. Rates of protein synthesis and degradation • Relative contributions of rates of protein synthesis and degradation can be summarized by – Where P is the concentration of protein, ksyn is the rate of synthesis, and kdeg is the rate of degradation © 2012 Pearson Education, Inc. Rates of protein synthesis and degradation • Rates of protein degradation are often expressed as the half-life of a protein • Enzymes with short half-lives change more dramatically in response to alterations in rate of synthesis than proteins with longer half-lives • Enzymes important in metabolic regulation tend to have short half-lives © 2012 Pearson Education, Inc. Ubiquitin Targets Proteins for Degradation by Proteasomes • Ubiquitin is a small protein containing 76 amino acids • It is joined to target proteins by a process involving three components – A ubiquitin-activating enzyme (E1) – A ubiquitin-conjugating enzyme (E2) – A substrate recognition protein, or ubiquitin ligase (E3) © 2012 Pearson Education, Inc. Figure 23-35 © 2012 Pearson Education, Inc. Proteasomes • Proteasomes are large protein-degrading structures, and the predominant proteases of the cytosol; they bind ubiquitin-labeled proteins and remove the ubiquitin • The proteins are then fed into the central channel of the proteasome and their peptide bonds are hydrolyzed in an ATP-dependent manner © 2012 Pearson Education, Inc. Protein degradation • Internal amino acid sequences called degrons target particular proteins for degradation • Certain N-terminal amino acids of proteins are sometimes targeted for ubiquitylation • Proteasomes also play a role in general mechanisms for eliminating defective proteins from cells © 2012 Pearson Education, Inc. SUMOylation • Proteins can also be modified by the addition of small ubiquitin-related modifiers (SUMOs) • SUMOylation alters protein stability, movement into and out of the nucleus, and regulation of transcription factor function © 2012 Pearson Education, Inc. Other means of degrading proteins • Lysosomes take up and degrade proteins by an infolding of the lysosomal membrane • Small vesicles are created that are internalized inside the lysosome and broken down there • This is microautophagy and the result is a slow continual recycling of amino acids © 2012 Pearson Education, Inc. A Summary of Eukaryotic Gene Regulation • There are five distinct levels of regulation possible – – – – – Genomic control Transcription control RNA processing and localization Translational control Posttranslational control © 2012 Pearson Education, Inc.