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• Key Concepts:

  • Bacteria regulate transcription in response to environmental change

  • Eukaryotic gene expression is regulated at many stages

  • Noncoding RNAs play multiple roles in controlling gene expression

  • Differential gene expression leads to different cell types in a multicellular organism

  • Cancer results from genetic changes affecting cell cycle control

• The fish in Figure 18.1 has eyes that can see equally well in both air and water

• The upper half of the eye is suited for aerial vision and the lower half for aquatic vision

• The cells of the two parts of the eye express a slightly different set of genes involved in vision

• Prokaryotic and eukaryotic cells regulate gene expression

• Bacteria regulate gene expression in response to environmental conditions

• Eukaryotes regulate gene expression through various mechanisms, including the role of RNA molecules

• Gene regulation is crucial for proper cell function

• Gene regulation plays a role in embryonic development and cancer

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• Each reaction in a metabolic pathway is catalyzed by a specific enzyme

• Genes coding for enzymes in a pathway are clustered together on the bacterial chromosome

• Transcription of these genes gives rise to one long mRNA molecule that codes for multiple polypeptides

• Grouping genes of related function into one transcription unit allows for coordinated control

• Bacteria respond to environmental change by regulating transcription

• Bacteria conserve resources by expressing only the genes needed by the cell

• Bacteria can adjust enzyme activity and production level in response to changes in the environment

• Regulation of gene expression in bacteria is described by the operon model

• E. coli synthesizes tryptophan through a three-step pathway

• Regulation of the tryptophan synthesis pathway is an example of how bacteria tune their metabolism to changing environments

• The operon model was discovered by François Jacob and Jacques Monod in 1961

Regulation of Gene Expression in the trp Operon

Main Ideas:

• The trp operon is a stretch of DNA that controls the production of enzymes for the tryptophan pathway.

• The trp operon can be switched off by a protein called the trp repressor, which binds to the operator and prevents transcription of the genes.

• The trp repressor is encoded by a regulatory gene called trpR, which is located separately from the trp operon.

• The binding of repressors to operators is reversible, and the duration of the repressor-bound state depends on the concentration of active repressor molecules.

• The trp repressor is an allosteric protein that can switch between active and inactive forms.

• Tryptophan acts as a corepressor, binding to the trp repressor and activating it to bind to the operator, turning the operon off.

• When tryptophan levels drop, the trp repressor proteins become inactive and dissociate from the operator, allowing transcription of the operon's genes to resume.

Supporting Details:

• The trp operon is one of many operons in the E. coli genome.

• The trp repressor is specific for the operator of the trp operon and does not affect other operons.

• The trp repressor is synthesized in the inactive form and has little affinity for the trp operator.

• Tryptophan molecules bind to the trp repressor at an allosteric site, changing it to the active form that can attach to the operator.

• Tryptophan functions as a corepressor, cooperating with the trp repressor to switch off the operon.

• As tryptophan accumulates, more tryptophan molecules associate with trp repressor molecules, leading to the shutdown of tryptophan pathway enzyme production.

• When tryptophan levels decrease, the trp repressor proteins become inactive, dissociate from the operator, and allow transcription of the operon's genes to resume.

Visual Skills:

• The trp operon is turned on when tryptophan is absent, and RNA polymerase can bind to the promoter and transcribe the genes.

• The trp operon is turned off when tryptophan is present, and the trp repressor protein binds to the operator, blocking transcription.

• Tryptophan acts as a corepressor, activating the trp repressor protein when it accumulates.

Note: The trp operon is an example of how gene expression can respond to changes in the cell's internal and external environment.

Page 4: Regulation of the lac operon in E. coli

• The gene for β-galactosidase (lacZ) is part of the lac operon, which includes two other genes coding for enzymes that function in the use of lactose.

  • The entire transcription unit is under the command of one main operator and promoter.

• The lac repressor protein, encoded by the regulatory gene lacI, can switch off the lac operon by binding to the lac operator.

  • The lac repressor is active by itself and binds to the operator, switching the lac operon off.

• The lac operon is regulated by the presence of a specific small molecule called an inducer.

  • The inducer for the lac operon is allolactose, an isomer of lactose formed in small amounts from lactose that enters the cell.

  • In the absence of lactose, the lac repressor is in its active shape and binds to the operator, silencing the genes of the lac operon.

  • If lactose is added to the cell's surroundings, allolactose binds to the lac repressor and alters its shape, preventing it from binding to the operator.

  • Without the lac repressor bound, the lac operon is transcribed into mRNA, and the enzymes for using lactose are made.

• The enzymes of the lac operon are referred to as inducible enzymes because their synthesis is induced by the presence of allolactose.

• The lac operon is an example of an inducible operon, which is usually off but can be stimulated to be on when a specific small molecule interacts with a regulatory protein.

Page 4: The lac operon in E. coli

• E. coli uses three enzymes to take up and metabolize lactose, which are encoded by the lac operon.

  • The first gene, lacZ, codes for β-galactosidase, which hydrolyzes lactose to glucose and galactose.

  • The second gene, lacY, codes for a permease, a membrane protein that transports lactose into the cell.

  • The third gene, lacA, codes for transacetylase, an enzyme that detoxifies other molecules entering the cell via the permease.

• The lac repressor protein is encoded by the gene lacI, which is adjacent to the lac operon.

Note: The lac operon in E. coli is regulated by the presence of lactose and allolactose, allowing the cell to efficiently produce the enzymes necessary for lactose metabolism.

Regulation of Gene Expression

Lac Operon Regulation

• cAMP controls the rate of transcription of the lac operon

• CRP (cAMP receptor protein) helps regulate other operons

• When glucose is plentiful and CRP is inactive, synthesis of enzymes for compounds other than glucose slows down

• Ability to catabolize other compounds enables cell survival without glucose

• Operons are switched on/off based on interactions of activator and repressor proteins with gene promoters

• Inducible enzymes function in catabolic pathways, produced only when nutrient is available

• Negative control of genes in trp and lac operons by active form of repressor protein

• Allolactose induces enzyme synthesis by freeing lac operon from negative effect of repressor

Positive Gene Regulation

• E. coli preferentially uses glucose when both glucose and lactose are present

• Only when lactose is present and glucose is scarce, E. coli uses lactose as an energy source and synthesizes enzymes for lactose breakdown

• E. coli senses glucose concentration and relays information to lac operon through interaction of allosteric regulatory protein (CRP) with cyclic AMP (cAMP)

• CRP binds to DNA and stimulates transcription of a gene

• Binding of cAMP to CRP increases affinity of RNA polymerase for lac promoter, stimulating gene expression

• Dual control of lac operon: negative control by lac repressor and positive control by CRP

• Lac repressor determines whether transcription occurs, CRP determines rate of transcription

Regulation of lac Operon in Different Conditions

• Lactose present, glucose scarce: abundant lac mRNA synthesized

• High level of cAMP activates CRP, which binds to promoter and increases RNA polymerase binding

• Lactose present, glucose present: little lac mRNA synthesized

• When glucose is present, cAMP is scarce, and CRP is unable to stimulate transcription significantly

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• Regulation of gene expression is essential for all organisms

• Differential gene expression allows for cell specialization in multicellular organisms

• Gene expression is the expression of different genes by cells with the same genome

• Abnormal gene expression can lead to imbalances and diseases, including cancer

• Gene expression in eukaryotic cells involves multiple stages, including transcription, RNA processing, translation, and protein processing

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• Chemical modifications to histones play a direct role in gene transcription

• Histone acetylation promotes transcription by opening up chromatin structure

• Methylation of histones can lead to the condensation of chromatin and reduced transcription

• DNA methylation can also regulate gene expression by turning genes on or off

• Gene expression in eukaryotes can be controlled at multiple stages, including transcription and chromatin structure

Page 8: Regulation of Transcription Initiation and Organization of a Typical Eukaryotic Gene

• Alterations in DNA methylation are seen in some cancers, affecting gene expression.

  • Enzymes that modify chromatin structure play a role in regulating transcription.

• Chromatin-modifying enzymes control gene expression by modifying DNA's ability to bind the transcription machinery.

• Transcription initiation in eukaryotes involves proteins that either facilitate or inhibit binding of RNA polymerase.

• Eukaryotic genes are organized with a transcription initiation complex assembling on the promoter sequence.

• DNA methylation patterns are maintained through cell divisions and can be passed on to daughter cells.

• Methylation patterns account for genomic imprinting in mammals, regulating expression of specific genes.

• Chromatin modifications can be passed along to future generations of cells, known as epigenetic inheritance.

• Epigenetic variations may explain why one identical twin acquires a genetically based disease while the other does not.

Figure 18.8: Structure of a Eukaryotic Gene and Its Transcript

• Each eukaryotic gene has a promoter where RNA polymerase binds and starts transcription.

• Control elements, located near or far from the promoter, regulate the initiation of transcription.

• Distal control elements can be grouped together as enhancers.

• The last exon of the gene contains a polyadenylation (poly-A) signal sequence.

• RNA processing involves addition of the 5' cap, addition of the poly-A tail, and splicing.

• Transcription may continue beyond the poly-A signal before terminating.

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• RNA polymerase II transcribes genes by synthesizing a primary RNA transcript (pre-mRNA)

  • RNA processing includes addition of a 5' cap and a poly-A tail, as well as splicing out of introns

• Eukaryotic genes have multiple control elements that bind to transcription factors and regulate transcription

• General transcription factors act at the promoter of all genes

• Specific transcription factors bind to control elements that may be close to or farther away from the promoter

• Enhancers are control elements located close to the promoter, while distal control elements are located further away, and groupings of distal control elements are called enhancers

• Specific transcription factors can strongly increase or decrease the rate of gene expression by binding to control elements in enhancers

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• Repressors can inhibit gene expression by binding directly to control element DNA or interfering with activator binding

• Activators and repressors can also affect chromatin structure, promoting or reducing transcription

• Combinatorial control of gene activation relies on the binding of activators to DNA control elements

• Despite the large number of genes, the number of different nucleotide sequences in control elements is small

• Each enhancer is composed of about ten control elements, each of which can bind to specific activators

Page 11: Regulation of Gene Expression

• Differential regulation of transcription in different cell types

  • Different combinations of control elements allow for differential regulation

  • Liver cells and lens cells as representative cell types

  • Each cell type contains a different group of activator proteins

• Importance of control element combinations in regulating transcription

  • Enhancer associated with a gene contains a particular combination of control elements

  • Presence of a single unique control element is not as important as the combination of control elements

  • Large number of combinations possible with a dozen control element sequences

Analyzing DNA Deletion Experiments

• Purpose of the experiment: to identify control elements that regulate the expression of the mPGES-1 gene

• Experimental setup

  • Three possible control elements in an enhancer region located 8-9 kilobases upstream of the mPGES-1 gene

  • DNA constructs with intact enhancer region and reporter gene

  • Three DNA constructs with one of the proposed control elements deleted

  • Introduction of DNA constructs into separate human cell cultures

• Measurement of reporter gene mRNA after 48 hours

• Data from the experiment

  • Diagrams showing intact DNA sequence and experimental DNA constructs

  • Bar graph showing the amount of reporter gene mRNA relative to the intact enhancer region

• Interpretation of the data

  1. Independent variable: presence or absence of control elements

     • Dependent variable: amount of reporter gene mRNA

     • Control treatment: intact enhancer region

  2. Data suggest that some of the possible control elements are actual control elements

  3. Deletion of certain control elements caused a reduction in reporter gene expression

     • Loss of a control element leads to a reduction in gene expression

  4. Deletion of certain control elements caused an increase in reporter gene expression

     • Loss of a control element leads to an increase in gene expression

Note: The note includes the main ideas from the transcript, with supporting details provided in sub-bullets. The note is organized into two sections: "Regulation of Gene Expression" and "Analyzing DNA Deletion Experiments."

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• Steroid hormones stimulate gene transcription by binding to control elements recognized by hormone-receptor complexes.

  • Estrogen activates genes that stimulate cell division in uterine cells.

• Signaling molecules like nonsteroid hormones and growth factors can indirectly control gene expression by triggering signal transduction pathways.

  • These pathways activate specific transcription factors.

• Coordinate regulation of gene expression is widespread and likely evolved early in evolutionary history.

• Chromosome conformation capture (3C) techniques allow researchers to identify regions of chromosomes that interact with each other in the nucleus.

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• Eukaryotic cells do not have operons like bacteria to coordinate gene expression.

• Coordinate gene expression in eukaryotes relies on specific combinations of control elements in dispersed genes.

• Chemical signals from outside the cell can trigger coordinate control of dispersed genes.

• Post-transcriptional regulation mechanisms fine-tune gene expression after transcription.

• RNA processing in the nucleus and export to the cytoplasm provide opportunities for regulating gene expression.

• Alternative RNA splicing produces different mRNA molecules from the same primary transcript.

• Chromatin loops extend from chromosomal territories into specific sites in the nucleus.

• Transcription factories are specialized areas in the nucleus where chromatin loops congregate.

• Genes that are not being expressed are located in the outer edges of the nucleus, while expressed genes are found in the interior region.

• Relocation of genes from chromosomal territories to transcription factories may be part of the process of preparing genes for transcription.

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• Nucleotide sequences in the untranslated region at the 3' end of mRNA can affect how long the mRNA remains intact

  • In an experiment, a sequence from a short-lived mRNA for a growth factor was transferred to the 3' end of a stable globin mRNA, resulting in quick degradation of the globin mRNA

• Other mechanisms that degrade or block expression of mRNA molecules have been discovered

  • These mechanisms involve newly discovered RNA molecules that regulate gene expression at multiple levels

• Eukaryotic polypeptides often need to be processed to yield functional protein molecules

  • For example, cleavage of pro-insulin forms the active hormone

  • Many proteins undergo chemical modifications to become functional

  • Regulatory proteins can be activated or inactivated by the addition of phosphate groups

  • Proteins destined for the cell surface acquire sugars

  • Proteins must be transported to target destinations in the cell to function

• Protein degradation is regulated to control the length of time each protein functions in the cell

  • Proteins marked for destruction have molecules of ubiquitin attached to them

  • Proteasomes recognize the ubiquitin-tagged proteins and degrade them

• Alternative RNA splicing can significantly expand the repertoire of a eukaryotic genome

  • Alternative splicing was proposed as an explanation for the low number of human genes counted when the human genome was sequenced

  • More than 90% of human protein-coding genes likely undergo alternative splicing, multiplying the number of possible human proteins

• Translation initiation and mRNA degradation are opportunities for regulating gene expression

  • Translation initiation can be blocked by regulatory proteins binding to specific sequences or structures within the untranslated region of mRNA

  • Global control of translation involves the activation or inactivation of protein factors required for translation initiation

  • mRNA life span in the cytoplasm determines the pattern of protein synthesis in a cell

  • Bacterial mRNA molecules have a short life span, allowing bacteria to quickly change their patterns of protein synthesis in response to environmental changes

  • mRNA molecules in multicellular eukaryotes typically survive for hours,