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11 Regulation of Gene Expression Chapter 11 Regulation of Gene Expression Key Concepts 11.1 Many Prokaryotic Genes Are Regulated in Operons 11.2 Eukaryotic Genes Are Regulated by Transcription Factors 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Chapter 11 Opening Question How does CREB regulate the expression of many genes? Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Gene expression can be precisely regulated at many different points: • Before or during transcription • Before or during translation • After translation Figure 11.1 Potential Points for the Regulation of Gene Expression Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Gene expression begins at the promoter. Regulatory proteins called transcription factors control gene activity: • Repressors prevent transcription (negative regulation) • Activators stimulate transcription (positive regulation) Figure 11.2 Positive and Negative Regulation Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Prokaryotes conserve energy and resources by making certain proteins only when they are needed. They can rapidly change expression levels as environmental conditions change. • Example: lactose catabolism in E. coli Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Uptake and metabolism of lactose involves three proteins: • β-galactoside permease—moves lactose into the cell • β-galactosidase—hydrolyses lactose • β-galactoside transacetylase—transfers acetyl groups to β-galactosides; role is unclear If E. coli is grown with glucose but no lactose, these enzymes are very low (basal level). Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons If cells are transferred to lactose medium, they begin making all 3 enzymes within 10 minutes. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons After lactose is added, the mRNA level increases before β-galactosidase begins to rise: Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons If lactose is removed, the mRNA level goes down. Response of the bacteria to lactose is at the level of transcription. Compounds that stimulate transcription of specific genes are called inducers, the genes are inducible genes. Genes that are expressed most the time at a constant rate are called constitutive genes. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The three genes for lactose enzymes form an operon: they are adjacent on the chromosome, share a promoter, and are transcribed together. The lac operon is very efficient but activity can be reduced when it is not needed—an example of transcriptional regulation. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The operon includes an operator—a short stretch of DNA near the promoter that controls transcription. Repressor proteins bind at the operator. • Inducible operons are turned off unless needed. • Repressible operons are turned on unless not needed. Figure 11.3 The lac Operon of E. coli Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The lac operon is inducible: the repressor prevents transcription until a β-galactoside predominates. A repressor protein is normally bound to the operator, blocking transcription. In the presence of lactose, the repressor detaches and allows RNA polymerase to initiate transcription. Figure 11.4 The lac Operon: An Inducible System (Part 1) Figure 11.4 The lac Operon: An Inducible System (Part 2) Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The repressor protein gene is constitutive (one that is always active), so the repressor is always present. It binds to the operator, but also has an allosteric binding site for the inducer (allolactose). When the inducer binds, the repressor changes shape and can no longer bind to the operator. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The lac repressor gene (lac i) is upstream of the lac operon. • It is a regulatory gene—it encodes a regulatory protein (transcription factor). Structural genes encode proteins that are not directly involved in gene regulation. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons A repressible operon is switched off when its repressor is bound to its operator. The repressor only binds in the presence of a corepressor. The corepressor causes the repressor to change shape and bind to the operator to inhibit transcription. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The trp operon for tryptophan synthesis is repressible. When tryptophan is present in adequate quantities, the cell can stop making enzymes for its synthesis. Tryptophan itself functions is the corepressor: it binds to the repressor, causing the repressor to bind to the trp operator to prevent transcription. Figure 11.5 The trp Operon: A Repressible System (Part 1) Figure 11.5 The trp Operon: A Repressible System (Part 2) Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Inducible system—a metabolic substrate (inducer) interacts with a regulatory protein (repressor); the repressor cannot bind and transcription proceeds. • Generally controls catabolic pathways. Repressible system—a metabolic product (corepressor) binds to a regulatory protein, which then binds to the operator and blocks transcription. • Generally controls anabolic pathways. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Transcription in prokaryotes can also be regulated by activator proteins that bind to DNA sequences at or near the promoter and promote transcription. Activators can regulate both inducible and repressible systems. Many genes and operons are controlled by more than one regulatory mechanism. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Two systems for regulating metabolic pathways: • Allosteric regulation of enzyme activity (feedback inhibition) • Regulation of transcription—slower, but results in greater savings of energy and resources Figure 11.6 Systems to Regulate a Metabolic Pathway Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Sigma factors can bind to RNA polymerase and direct it to specific promoters. Global gene regulation: genes that encode proteins with related functions may be at different locations but have the same promoter sequence—thus they can be expressed at the same time. • RNA polymerase is directed to the promoter in each case by a specific sigma factor. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons A virus injects its genetic material into a host cell, often turning it into a virus factory: Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Viruses are not cells and are dependent on living cells to reproduce. Viral genomes may be double- or singlestranded DNA or RNA. Lytic life cycle: the host cell immediately starts producing new viral particles (virions), which are released as the cell breaks open, or lyses Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons Lysogenic life cycle: viral genome is incorporated into host cell genome and is replicated along with host genome The virus may survive in this way for many host cell generations. An environmental signal can cause the host cell to start producing virions, and the viral reproductive cycle enters the lytic phase. Concept 11.1 Many Prokaryotic Genes Are Regulated in Operons The lytic cycle has two stages: • Early—viral genes adjacent to a promoter that binds host RNA polymerase are transcribed. § Early genes encode proteins that shut down expression of host genes, stimulate viral genome replication, and activate transcription of viral late genes. • Late—viral late genes encode viral capsid proteins and enzymes that lyse the host cell. Figure 11.7 A Gene Regulation Strategy for Viral Reproduction Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Eukaryotic cells must also regulate expression of their genes. Some are constitutive; others are inducible. This is especially important in multicellular organisms. There are significant differences between prokaryotes and eukaryotes, which generally reflect the greater complexity of eukaryotes. Table 11.1 Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors and DNA Changes Eukaryotic promoters are DNA regions where RNA polymerase binds and initiates transcription. A common core sequence is the TATA box (rich in A–T base pairs). General transcription factors bind to the core promoter, then RNA polymerase II binds and initiates transcription. Figure 11.8 The Initiation of Transcription in Eukaryotes Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Other promoter sequences are specific to a few genes and are recognized by specific transcription factors. Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors DNA sequences that bind activators are enhancers, those that bind repressors are silencers. The combination of factors present determines whether transcription is initiated. Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Transcription factors recognize particular nucleotide sequences. • Example: NFATs (nuclear factors of activated T cells) control genes in the immune system. NFAT proteins bind to a recognition sequence by hydrogen bonding and hydrophobic interactions. There is an induced fit between the NFAT and the DNA, and the protein undergoes a conformational change. Figure 11.9 A Transcription Factor Protein Binds to DNA Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors In multicellular organisms, all differentiated cells contain the entire genome; their specific characteristics arise from differential gene expression. Figuring out how to get undifferentiated cells, such as fibroblasts, to differentiate into specialized cells, such as neurons, may prove to be effective in treating many diseases, such as Alzheimer’s, which involves degeneration of neurons. Figure 11.10 Expression of Specific Transcription Factors Turns Fibroblasts into Neurons (Part 1) Figure 11.10 Expression of Specific Transcription Factors Turns Fibroblasts into Neurons (Part 2) Figure 11.10 Expression of Specific Transcription Factors Turns Fibroblasts into Neurons (Part 3) Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Coordination of gene expression: Even if they are far apart, genes can share regulatory sequences that bind the same transcription factors. • Example: plant response to drought—the scattered stress response genes all have a regulatory sequence called the dehydration response element. § The resulting proteins help the plant conserve water and protect against freezing or excess salt. Figure 11.11 Coordinating Gene Expression Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Human immunodeficiency virus (HIV) has a complex life cycle. It infects only cells in the immune system that have the surface receptor CD4. The virion is enclosed in a membrane from the previous host cell, which fuses with the new host cell’s membrane. Figure 11.12 The Reproductive Cycle of HIV Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors HIV is a retrovirus: its genome is singlestranded RNA. After infection, reverse transcriptase makes a DNA strand that is complementary to the RNA, while at the same time degrading the RNA and making a second DNA strand. The resulting double-stranded DNA is integrated into the host’s chromosome. The integrated viral DNA is called a provirus. Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors The provirus resides permanently in the host chromosome and can be inactive for years. Transcription of the viral DNA may be initiated, but host cell proteins (termination factors) prevent the RNA from elongating. Figure 11.13 Regulation of Transcription by HIV Concept 11.2 Eukaryotic Genes Are Regulated by Transcription Factors Under some circumstances, a viral gene encodes Tat (Transactivator of transcription), which allows RNA polymerase to transcribe the viral genome. The rest of the viral reproductive cycle is then able to proceed. After discovery of this mechanism, researchers found that many eukaryotic genes are regulated at the level of transcription elongation. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Gene transcription can also be regulated by reversible alterations to DNA or chromosomal proteins—epigenetic changes. Although base sequences are not changed, these alterations can be passed on to daughter cells. One type of alteration is chromatin remodeling. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin DNA is packaged into nucleosomes— positively charged histone proteins around which DNA is wound: Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin The “tails” have positively charged amino acids, especially lysine. There is strong ionic attraction between the positive histone proteins and DNA (negatively charged because of phosphate groups). Thus, nucleosomes can make DNA physically inaccessible to RNA polymerase. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Histone acetyltransferases can add acetyl groups to the positive amino acids and neutralize them: Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Reducing positive charges on the histone tails reduces affinity of the histones for the DNA, loosening the compact nucleosome. Histone acetylation promotes both transcription initiation and elongation. Histone acetylases can be recruited to promoters by transcription factors, such as CREB. Figure 11.14 Epigenetic Remodeling of Chromatin for Transcription Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Histone deacetylase removes acetyl groups from histones, repressing transcription. Histones can also be modified by methylation (addition of methyl groups) and phosphorylation (addition of a phosphate groups). All these effects are reversible, so transcriptional activity may be determined by varying patterns of histone modification. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Cytosines in DNA are chemically modified by addition of a methyl group (—CH3) to form 5methylcytosine, catalyzed by DNA methyltransferase. In mammals this usually occurs on cytosines (C) that are adjacent to guanines (G). CpG sites are nearly always methylated. Promoters have regions rich in C and G, called CpG islands. Figure 11.15 DNA Methylation: An Epigenetic Change (Part 1) Figure 11.15 DNA Methylation: An Epigenetic Change (Part 2) Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Methylated DNA binds proteins involved in repression of transcription; heavily methylated genes tend to be inactive (silenced). DNA methylation is usually a stable, long-term silencing mechanism. When DNA replicates, maintenance methyltransferase catalyzes formation of 5methylcytosine in the new DNA strand. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin But methylation is reversible, so methylation patterns can be altered. Demethylase catalyzes removal of methyl groups from cytosine. Genes whose products are needed are kept unmethylated, and their associated histones acetylated. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Two kinds of chromatin are visible during interphase: • Euchromatin—diffuse and light-staining; unmethylated; DNA is available for transcription • Heterochromatin—condensed, darkstaining, methylated; contains genes not transcribed Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin A type of heterochromatin is the inactive X chromosome in mammals. Early in the development of a female, one of the X chromosomes becomes heterochromatic and transcriptionally inactive. Which X becomes inactive is random; in one embryonic cell the paternal X might be inactivated, but in a neighboring cell the maternal X might be inactivated. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Example of a phenotype caused by X chromosome inactivation: the tortoiseshell cat. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin The inactive X is identifiable in the nucleus as a heterochromatic Barr body. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Methylation patterns are stable and can be passed from one generation to the next. But studies of monozygotic twins show they can be altered. Comparison of DNA in such twin pairs shows that in 3-year-olds, DNA methylation patterns are virtually the same. By age 50, when twins have been living in different environments—the patterns are different, and different genes are expressed. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin These studies show that environment plays an important role in epigenetic modifications, and thus gene regulation. Environmental factors that may be involved include chemicals in tobacco smoke, dietary components, and stress. Concept 11.3 Gene Expression Can Be Regulated via Epigenetic Changes to Chromatin Genomic imprinting: In mammals, DNA methylation patterns in sperm differs from that in eggs at about 200 genes. A given gene in this group may be methylated in eggs but unmethylated in sperm. The offspring would inherit one methylated copy and one unmethylated copy. Most imprinted genes are involved with embryonic development. Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Eukaryotic gene expression can be regulated after transcription. Different mRNAs can be made from one gene by alternative splicing: different combinations of introns are spliced together. Figure 11.16 Alternative Splicing Results in Different Mature mRNAs and Proteins Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Examples of alternative splicing: • The HIV genome encodes nine proteins, but is transcribed as a single pre-mRNA. • In Drosophila, sex is determined by the Sxl gene, which has four exons. Splicing generates different exon combinations in males and females. Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Some noncoding regions of DNA are transcribed into microRNA (miRNA). miRNA was discovered in C. elegans during research on genes that control larval development stages: • lin-14 encodes a transcription factor for genes that control events in the 1st stage. • lin-4 negatively regulates lin-14, so cells can progress to the next stage. It encodes an miRNA that inhibits lin-14 expression by binding to its mRNA. Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Many miRNAs have now been described. Once transcribed, they are folded and processed, then guided to a target mRNA by protein complexes. Translation is thus inhibited and the mRNA is degraded. This mechanism is found in most eukaryotes, indicating its importance and that it is evolutionarily ancient. Figure 11.17 mRNA Degradation Caused by MicroRNAs Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Amounts of proteins and their mRNAs in a cell are not consistently related. Protein concentrations can be adjusted by regulating mRNA translation or altering how long proteins persist. Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Three ways to regulate mRNA translation: • Inhibit translation with miRNAs • Modify the 5′ cap of mRNA. If the cap is unmodified mRNA is not translated. • Translational repressor proteins bind to mRNAs and prevent their attachment to ribosomes. § Example: ferritin binds free Fe2+ ions; when it is low, a repressor binds to the ferritin mRNA. Figure 11.18 A Repressor of Translation Concept 11.4 Eukaryotic Gene Expression Can Be Regulated after Transcription Protein content of any cell at a given time is a function of synthesis and degradation. Proteins can be targeted for destruction when an enzyme attaches a ubiquitin to a lysine. Other ubiquitins then attach to form a protein– polyubiquitin complex. The complex binds to a proteasome where the polyubiquitin is removed and the protein is digested by proteases. Figure 11.19 A Proteasome Breaks Down Proteins Answer to Opening Question The CREB family of transcription factors can activate or repress gene expression by binding to the cAMP response element (CRE) sequence found in the promoter region of many genes. CREB binding is essential in many organs, including the brain, and plays a role in drug and alcohol addiction. Figure 11.20 An Explanation for Alcoholism? Answer to Opening Question CREB also has a role in long-term memory. When animals learn a task, imaging studies show CRE-containing genes becoming active in the hippocampus. CREB provides insights into the molecular biology of memory, linking learning to the regulation of gene expression.