Regulation of Gene Expression

Regulation of Gene Expression
Chapter 18 Regulation of Gene Expression Modified by Bronwyn Scott Nov. 2008

Overview: Conducting the Genetic Orchestra
Prokaryotes & eukaryotes alter gene expression in response to their changing environment In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types Gene expression in eukaryotes and bacteria is often regulated at the transcription stage. Both prokaryotes and eukaryotes alter their patterns of gene expression in response to changes in environmental conditions. In multicellular eukaryotes, each cell type contains the same genome but expresses a different subset of genes. During development, gene expression must be carefully regulated to ensure that the right genes are expressed only at the correct time and in the correct place. Gene expression in eukaryotes and bacteria is often regulated at the transcription stage. Control of other levels of gene expression is also important. RNA molecules play many roles in regulating eukaryotic gene expressions. Disruptions in gene regulation can lead to cancer.

Gene expression in bacteria is controlled by the operon model
Concept 18.1: Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the products needed by that cell A cell can regulate the production of enzymes by feedback inhibition or by gene regulation Gene expression in bacteria is controlled by the operon model Both prokaryotes and eukaryotes alter their patterns of gene expression in response to changes in environmental conditions. In multicellular eukaryotes, each cell type contains the same genome but expresses a different subset of genes. During development, gene expression must be carefully regulated to ensure that the right genes are expressed only at the correct time and in the correct place. Gene expression in eukaryotes and bacteria is often regulated at the transcription stage. Control of other levels of gene expression is also important. RNA molecules play many roles in regulating eukaryotic gene expressions. Disruptions in gene regulation can lead to cancer. Concept 18.1 Bacteria often respond to environmental change by regulating transcription. Natural selection favors bacteria that express only those genes whose products are needed by the cell. A bacterium in a tryptophan-rich environment that stops producing tryptophan conserves its resources. Metabolic control occurs on two levels. First, cells can adjust the activity of enzymes already present. This may happen by feedback inhibition, in which the activity of the first enzyme in a pathway is inhibited by the pathway’s end product. Feedback inhibition, typical of anabolic (biosynthetic) pathways, allows a cell to adapt to short-term fluctuations in the supply of a needed substance. Second, cells can vary the number of specific enzyme molecules they make by regulating gene expression. Genes of the bacterial genome may be switched on or off by changes in the metabolic status of the cell. The basic mechanism for the control of gene expression in bacteria, known as the operon model, was described by Francois Jacob and Jacques Monod in 1961.

(a) Regulation of enzyme activity (b) Regulation of enzyme production
Fig. 18-2 Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Regulation of gene expression Enzyme 2 trpC gene trpB gene Figure 18.2 Regulation of a metabolic pathway Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production

Operons: The Basic Concept
A cluster of functionally related genes can be under coordinated control by a single on-off “switch” The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control Escherichia coli synthesizes tryptophan from a precursor molecule in a series of steps, with each reaction catalyzed by a specific enzyme. The five genes coding for these enzymes are clustered together on the bacterial chromosome as a transcription unit, served by a single promoter. Transcription gives rise to one long mRNA molecule that codes for all five enzymes in the tryptophan pathway. The mRNA is punctuated with start and stop codons that signal where the coding sequence for each polypeptide begins and ends. A key advantage of grouping genes with related functions into one transcription unit is that a single on-off switch can control a cluster of functionally related genes. In other words, these genes are under coordinate control. When an E. coli cell must make tryptophan for itself, all the enzymes are synthesized at one time. The switch is a segment of DNA called an operator. The operator, located between the promoter and the enzyme-coding genes, controls the access of RNA polymerase to the genes. The operator, the promoter, and the genes they control constitute an operon. The trp operon (trp for tryptophan) is one of many operons in the E. coli genome. By itself, an operon is turned on. RNA polymerase can bind to the promoter and transcribe the genes.

The operon can be switched off by a protein repressor
The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase The repressor is the product of a separate regulatory gene The operon can be switched off by a protein called the trp repressor. The repressor binds to the operator, blocks attachment of RNA polymerase to the promoter, and prevents transcription of the operon’s genes. Each repressor protein recognizes and binds only to the operator of a particular operon. The trp repressor is the product of a regulatory gene called trpR, which is located at some distance from the operon it controls and has its own promoter. Regulatory genes are transcribed continuously at slow rates, and a few trp repressor molecules are always present in an E. coli cell.

Polypeptide subunits that make up enzymes for tryptophan synthesis
Fig. 18-3a trp operon Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Regulatory gene Operator Start codon Stop codon 3 mRNA 5 mRNA RNA polymerase 5 E D C B A Protein Inactive repressor Polypeptide subunits that make up enzymes for tryptophan synthesis Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes (a) Tryptophan absent, repressor inactive, operon on

For example, E. coli can synthesize the amino acid tryptophan
The repressor can be in an active or inactive form, depending on the presence of other molecules A corepressor is a molecule that cooperates with a repressor protein to switch an operon off For example, E. coli can synthesize the amino acid tryptophan Why is the trp operon not switched off permanently? First, binding by the repressor to the operator is reversible. An operator vacillates between two states, with and without a repressor bound to it. Second, repressors contain allosteric sites that change shape depending on the binding of other molecules. The trp repressor has two shapes: active and inactive. In the case of the trp operon, when concentrations of tryptophan in the cell are high, some tryptophan molecules bind as a corepressor to the repressor protein. This activates the repressor and turns the operon off. At low levels of tryptophan, most of the repressors are inactive, and the operon is transcribed. As tryptophan accumulates, more tryptophan molecules associate with trp repressor molecules, which bind to the trp operator and shut down production of the enzymes of the tryptophan pathway.

(b) Tryptophan present, repressor active, operon off
Fig. 18-3b-1 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes (b) Tryptophan present, repressor active, operon off

(b) Tryptophan present, repressor active, operon off
Fig. 18-3b-2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Figure 18.3 The trp operon in E. coli: regulated synthesis of repressible enzymes (b) Tryptophan present, repressor active, operon off

By default the trp operon is on and the genes for tryptophan synthesis are transcribed
When tryptophan is present, it binds to the trp repressor protein, which turns the operon off The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

The trp operon is a repressible operon
Repressible and Inducible Operons: Two Types of Negative Gene Regulation A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The trp operon is an example of a repressible operon, one that is inhibited when a specific small molecule (tryptophan) binds allosterically to a regulatory protein. In contrast, an inducible operon is stimulated or induced when a specific small molecule interacts with a regulatory protein. In inducible operons, the regulatory protein is active (inhibitory) as synthesized, and the operon is off. Allosteric binding by an inducer molecule makes the regulatory protein inactive, and the operon is turned on.

The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose By itself, the lac repressor is active and switches the lac operon off A molecule called an inducer inactivates the repressor to turn the lac operon on

(a) Lactose absent, repressor active, operon off
Fig. 18-4a Regulatory gene Promoter Operator DNA lacI lacZ No RNA made 3 mRNA RNA polymerase 5 Figure 18.4 The lac operon in E. coli: regulated synthesis of inducible enzymes The lac operon contains a series of genes that code for enzymes that play a major role in the hydrolysis and metabolism of lactose (milk sugar). In the absence of lactose, this operon is off because an active repressor binds to the operator and prevents transcription. Lactose metabolism begins with hydrolysis of lactose into its component monosaccharides, glucose and galactose. This reaction is catalyzed by the enzyme ß-galactosidase. Only a few molecules of -galactosidase are present in an E. coli cell grown in the absence of lactose. Active repressor Protein (a) Lactose absent, repressor active, operon off

Inducers inactivate repressors
lac operon DNA lacI lacZ lacY lacA RNA polymerase 3 mRNA mRNA 5 5 -Galactosidase Permease Transacetylase Protein Figure 18.4 The lac operon in E. coli: regulated synthesis of inducible enzymes If lactose is added to the bacterium’s environment, the number of ß-galactosidase molecules increases by a thousandfold within 15 minutes. The gene for ß-galactosidase is part of the lac operon, which includes two other genes coding for enzymes that function in lactose metabolism. The regulatory gene, lacI, located outside the operon, codes for an allosteric repressor protein that can switch off the lac operon by binding to the operator. Unlike the trp operon, the lac repressor is active all by itself, binding to the operator and switching the lac operon off. An inducer inactivates the repressor. When lactose is present in the cell, allolactose, an isomer of lactose, binds to the repressor. This inactivates the repressor, and the lac operon can be transcribed. Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on If lactose is added to the bacterium’s environment, the number of ß-galactosidase molecules increases by a thousandfold within 15 minutes.

Repressible enzymes Repressible enzymes usually function in anabolic pathways; Which synthesize essential end products from raw materials By suspending production of an end product when it is already present in sufficient quantities, the cell can allocate its organic precursors and energy for other uses.

Inducible enzymes Inducible enzymes usually function in catabolic pathways; Which break down a nutrient to simplier molecules Their synthesis is induced by a chemical signal By producing the appropriate enzymes only when the nutrient is available, the cell avoids wasting energy and precursors making proteins that are not needed

Negative vs. Positive Control
Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor protein. Gene regulation is said to be positive only when a regulatory protein interacts directly with the genome Both repressible and inducible operons demonstrate negative control of genes because active repressors switch off the active form of the repressor protein. Positive gene control occurs when a protein molecule interacts directly with the genome to switch transcription on. The lac operon is an example of positive gene regulation.

Positive Gene Regulation
When glucose and lactose are both present, E. coli prefers glucose. When glucose levels are low, cyclic AMP (cAMP) accumulates in the cell. The regulatory protein catabolite activator protein (CAP) is an activator of transcription. When cAMP is abundant, it binds to CAP, and the regulatory protein assumes its active shape and can bind to a specific site at the upstream end of the lac promoter. The attachment of CAP to the promoter increases the affinity of RNA polymerase for the promoter, directly increasing the rate of transcription. Thus, this mechanism qualifies as positive regulation. If glucose levels in the cell rise, cAMP levels fall. Without cAMP, CAP detaches from the operon. The lac operon is transcribed but at a low level. The lac operon is under dual control: negative control by the lac repressor and positive control by CAP. The state of the lac repressor (with or without bound allolactose) determines whether or not the lac operon’s genes are transcribed. The state of CAP (with or without bound cAMP) controls the rate of transcription if the operon is repressor-free. The operon has both an on-off switch and a volume control.

(a) Lactose present, glucose scarce (cAMP level
Fig. 18-5 Promoter Operator DNA lacI lacZ CAP-binding site RNA polymerase binds and transcribes Active CAP cAMP Inactive lac repressor Inactive CAP Allolactose (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Promoter Operator DNA lacI lacZ Figure 18.5 Positive control of the lac operon by catabolite activator protein (CAP) CAP-binding site RNA polymerase less likely to bind Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

CAP - catabolite activator protein
CAP works on several operons that encode enzymes used in catabolic pathways. It affects the expression of more than 100 E. coli genes. If glucose is present & CAP is inactive, then the synthesis of enzymes that catabolize other compounds is slowed. If glucose levels are low & CAP is active, then the genes that produce enzymes that catabolize whichever other fuel is present are transcribed at high levels. When cAMP is abundant, it binds to CAP, and the regulatory protein assumes its active shape and can bind to a specific site at the upstream end of the lac promoter. The attachment of CAP to the promoter increases the affinity of RNA polymerase for the promoter, directly increasing the rate of transcription. Thus, this mechanism qualifies as positive regulation. If glucose levels in the cell rise, cAMP levels fall. Without cAMP, CAP detaches from the operon. The lac operon is transcribed but at a low level. The lac operon is under dual control: negative control by the lac repressor and positive control by CAP. The state of the lac repressor (with or without bound allolactose) determines whether or not the lac operon’s genes are transcribed. The state of CAP (with or without bound cAMP) controls the rate of transcription if the operon is repressor-free. The operon has both an on-off switch and a volume control.

Concept 18.2: Eukaryotic gene expression can be regulated at any stage
All organisms must regulate which genes are expressed at any given time In multicellular organisms gene expression is essential for cell specialization Like unicellular organisms, the tens of thousands of genes in the cells of multicellular eukaryotes are continuously turned on and off in response to signals from their internal and external environments. Gene expression must be controlled on a long-term basis during cellular differentiation, the divergence in form and function as cells in a multicellular organism specialize.

Differential Gene Expression
Almost all the cells in an organism are genetically identical Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome Errors in gene expression can lead to diseases including cancer Gene expression is regulated at many stages A typical human cell probably expresses about 20% of its genes at any given time. Highly specialized cells, such as nerves or muscles, express only a tiny fraction of their genes. Although all the cells in an organism contain an identical genome, the subset of genes expressed in the cells of each type is unique. The differences between cell types are due to differential gene expression, the expression of different genes by cells with the same genome. The genomes of eukaryotes may contain tens of thousands of genes. For quite a few species, only a small amount of the DNA—1.5% in humans—codes for protein. Of the remaining DNA, a very small fraction consists of genes for rRNA and tRNA. The rest of the DNA either codes for RNA products, such as tRNAs, or isn’t transcribed. Problems with gene expression and control can lead to imbalance and disease, including cancer. Our understanding of the mechanisms that control gene expression in eukaryotes has been enhanced by new research methods, including advances in DNA technology. In all organisms, the expression of specific genes is most commonly regulated at transcription, often in response to signals coming from outside the cell. For this reason, the term gene expression is often equated with transcription. With their greater complexity, eukaryotes have opportunities for controlling gene expression at additional stages.

Signal NUCLEUS Chromatin Chromatin modification DNA Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Tail Cap mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Figure 18.6 Stages in gene expression that can be regulated in eukaryotic cells Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function

Regulation of Chromatin Structure
Genes within highly packed heterochromatin are usually not expressed A gene’s location relative to nucleosomes and to attachment sites to the chromosome scaffold or nuclear lamina can affect transcription. Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression The DNA of eukaryotic cells is packaged with proteins in a complex called chromatin. The location of a gene’s promoter relative to nucleosomes and to the sites where the DNA attaches to the chromosome scaffold or nuclear lamina can affect whether the gene is transcribed. Genes of densely condensed heterochromatin are usually not expressed, presumably because transcription proteins cannot reach the DNA. A gene’s location relative to nucleosomes and to attachment sites to the chromosome scaffold or nuclear lamina can affect transcription. Histone modifications regulate gene transcription. Chemical modifications of the histones and DNA of chromatin play a key role in chromatin structure and gene expression.

These histone tails are accessible to various modifying enzymes, which catalyze the addition or removal of specific chemical groups. Histone tails Amino acids available for chemical modification DNA double helix (a) Histone tails protrude outward from a nucleosome Figure 18.7 A simple model of histone tails and the effect of histone acetylation The N-terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome. Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

Histone Modifications
In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This process loosens chromatin structure, thereby promoting the initiation of transcription The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin Histone acetylation (addition of an acetyl group, —COCH3) and deacetylation appear to play a direct role in the regulation of gene transcription. Acetylated histones grip neighboring nucleosomes less tightly, providing easier access for transcription proteins in this region. Some of the enzymes responsible for acetylation or deacetylation are associated with or are components of transcription factors that bind to promoters. Thus, histone acetylation enzymes may promote the initiation of transcription not only by modifying chromatin structure but also by binding to and recruiting components of the transcription machinery. Several other chemical groups, such as methyl and phosphate groups, can be reversibly attached to amino acids in histone tails. The attachment of methyl groups (—CH3) to histone tails leads to condensation of chromatin. The addition of a phosphate group (phosphorylation) to an amino acid next to a methylated amino acid has the opposite effect.

The histone code hypothesis proposes that specific combinations of modifications help determine chromatin configuration and influence transcription The recent discovery that modifications to histone tails can affect chromatin structure and gene expression has led to the histone code hypothesis. This hypothesis proposes that specific combinations of modifications, rather than the overall level of histone acetylation, determine chromatin configuration. Chromatin configuration in turn influences transcription.

DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species DNA methylation can cause long-term inactivation of genes in cellular differentiation In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development While some enzymes methylate the tails of histone proteins, other enzymes methylate certain bases in DNA itself. The DNA of most plants, animals, and fungi has methylated bases, usually cytosine. Inactive DNA is generally highly methylated compared to DNA that is actively transcribed. For example, the inactivated mammalian X chromosome in females is heavily methylated. Genes are usually more heavily methylated in cells where they are not expressed. Demethylating certain inactive genes turns them on. DNA methylation proteins recruit histone deacetylation enzymes, providing a mechanism by which DNA methylation and histone deacetylation cooperate to repress transcription. In some species, DNA methylation is responsible for the long-term inactivation of genes during cellular differentiation. Once methylated, genes usually stay that way through successive cell divisions in a given individual. Methylation enzymes recognize sites on one strand that are already methylated and correctly methylate the daughter strand after each round of DNA replication. This methylation pattern accounts for genomic imprinting, in which methylation turns off either the maternal or paternal alleles of certain genes at the start of development.

Epigenetic Inheritance
Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance The chromatin modifications just discussed do not alter the DNA sequence, and yet they may be passed along to future generations of cells. Inheritance of traits by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance. Researchers are amassing more and more evidence for the importance of epigenetic information in the regulation of gene expression. Epigenetic variations may explain why one identical twin acquires a genetically based disease, such as schizophrenia, while another does not, despite their identical genomes. Enzymes that modify chromatin structure are integral parts of the cell’s machinery for regulating transcription.

Regulation of Transcription Initiation
Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery A cluster of proteins called a transcription initiation complex assembles on the promoter sequence at the upstream end of the gene. Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more available or less available for transcription. A cluster of proteins called a transcription initiation complex assembles on the promoter sequence at the upstream end of the gene. One component, RNA polymerase II, transcribes the gene, synthesizing a primary RNA transcript or pre-mRNA. RNA processing includes enzymatic addition of a 5 cap and a poly-A tail, as well as splicing out of introns to yield a mature mRNA.

Organization of a Typical Eukaryotic Gene
Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types Multiple control elements are associated with most eukaryotic genes. Control elements are noncoding DNA segments that regulate transcription by binding certain proteins. These control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types.

(distal control elements)
Fig Poly-A signal sequence Enhancer (distal control elements) Proximal control elements Termination region Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Primary RNA transcript Exon Intron Exon Intron Exon Cleaved 3 end of primary transcript 5 RNA processing Intron RNA Poly-A signal Figure 18.8 A eukaryotic gene and its transcript Coding segment mRNA 3 Start codon Stop codon 5 Cap 5 UTR 3 UTR Poly-A tail

The Roles of Transcription Factors
To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors. General transcription factors are essential for the transcription of all protein-coding genes. Only a few general transcription factors independently bind a DNA sequence such as the TATA box within the promoter. Others are involved in protein-protein interactions, binding each other and RNA polymerase II. Only when the complete initiation complex has been assembled can the polymerase begin to move along the DNA template strand to produce a complementary strand of RNA. The interaction of general transcription factors and RNA polymerase II with a promoter usually leads to only a slow rate of initiation and the production of few RNA transcripts. In eukaryotes, high levels of transcription of particular genes depend on the interaction of control elements with specific transcription factors.

Enhancers and Specific Transcription Factors
Proximal control elements are located close to the promoter Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron Some control elements, named proximal control elements, are located close to the promoter. Distal control elements, enhancers, may be thousands of nucleotides away from the promoter or even downstream of the gene or within an intron. A given gene may have multiple enhancers, each active at a different time or in a different cell type or location in the organism. Interactions between enhancers and specific transcription factors called activators or repressors are important in controlling gene expression.

Animation: Initiation of Transcription
An activator is a protein that binds to an enhancer and stimulates transcription of a gene Bound activators cause mediator proteins to interact with proteins at the promoter An activator is a protein that binds to an enhancer to stimulate transcription of a gene. Protein-mediated bending of DNA brings bound activators in contact with a group of mediator proteins that interact with proteins at the promoter. These interactions help assemble and position the initiation complex on the promoter. Hundreds of transcription activators have been discovered in eukaryotes. Two common structural elements characterize a large number of activator proteins: a DNA-binding domain and one or more activation domains. Activation domains bind other regulatory proteins or components of the transcription machinery to facilitate transcription. Animation: Initiation of Transcription

Promoter Activators Gene DNA Enhancer
Fig Promoter Activators Gene DNA Distal control element Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II Figure 18.9 A model for the action of enhancers and transcription activators RNA polymerase II Transcription initiation complex RNA synthesis

Some transcription factors function as repressors, inhibiting expression of a particular gene
Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription Recruitment of chromatin-modifying proteins seems to be the most common mechanism of repression in eukaryotes. Eukaryotic genes also have repressors to inhibit the expression of a specific gene. Eukaryotic repressors can inhibit gene expression by blocking the binding of activators to their control elements or to components of the transcription machinery. Other repressors bind directly to control-element DNA, turning off transcription even in the presence of activators. Some activators and repressors act indirectly to influence chromatin structure. Some activators recruit proteins that acetylate histones near the promoters of specific genes, promoting transcription. Some repressors recruit proteins that deacetylate histones, reducing transcription or silencing the gene. Recruitment of chromatin-modifying proteins seems to be the most common mechanism of repression in eukaryotes.

Combinatorial Control of Gene Activation
The number of nucleotide sequences found in control elements is surprisingly small: about a dozen. Different combinations of control elements allows fine regulation of transcription with a small set of control elements. A particular combination of control elements can activate transcription only when the appropriate activator proteins are present The number of nucleotide sequences found in control elements is surprisingly small: about a dozen. On average, each enhancer is composed of about ten control elements, each of which can bind to only one or two specific transcription factors. The particular combination of control elements in an enhancer may be more important than the presence of a single unique control element in regulating transcription of the gene. Even with only a dozen control element sequences, a large number of combinations are possible. A particular combination of control elements is able to activate transcription only when the appropriate activator proteins are present, such as at a precise time during development or in a particular cell type. The use of different combinations of control elements allows fine regulation of transcription with a small set of control elements.

Enhancer Promoter Albumin gene Control elements Crystallin gene
LIVER CELL NUCLEUS LENS CELL NUCLEUS Available activators Available activators Albumin gene not expressed Figure Cell type–specific transcription Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed (a) Liver cell (b) Lens cell

Coordinately Controlled Genes in Eukaryotes
Unlike the genes of a prokaryotic operon, each of the coordinately controlled eukaryotic genes has a promoter and control elements These genes can be scattered over different chromosomes, but each has the same combination of control elements Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes In prokaryotes, coordinately controlled genes are often clustered into an operon with a single promoter and other control elements upstream. The genes of the operon are transcribed into a single mRNA and translated together. In contrast, very few eukaryotic genes are organized this way. Recent studies of the genomes of several eukaryotic species have found that some co-expressed genes are clustered near one other on the same chromosome. Each gene in these clusters has its own promoter and is individually transcribed. The coordinate regulation of clustered genes in eukaryotic cells is thought to involve changes in the chromatin structure that make the entire group of genes either available or unavailable for transcription. In other cases, including 15% of nematode genes, several related genes share a promoter and are transcribed into a single pre-mRNA, which is then processed into separate mRNAs. More commonly, genes coding for the enzymes of a metabolic pathway are scattered over different chromosomes. Coordinate gene expression in eukaryotes depends on the association of a specific control element or combination of control elements with every gene of a dispersed group. A common group of transcription factors binds to all the genes in the group, promoting simultaneous gene transcription. For example, a steroid hormone enters a cell and binds to a specific receptor protein in the cytoplasm or nucleus, forming a hormone–receptor complex that serves as a transcription activator. Every gene whose transcription is stimulated by that steroid hormone has a control element recognized by that hormone–receptor complex. Other signal molecules control gene expression indirectly by triggering signal-transduction pathways that lead to activation of transcription. The principle of coordinate regulation is the same: Genes with the same control elements are activated by the same chemical signals. Systems for coordinating gene regulation probably arose early in evolutionary history and evolved by the duplication and distribution of control elements within the genome.

Mechanisms of Post-Transcriptional Regulation
Transcription alone does not account for gene expression Regulatory mechanisms can operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes Gene expression may be blocked or stimulated by any post-transcriptional step. By using regulatory mechanisms that operate after transcription, a cell can rapidly fine-tune gene expression in response to environmental changes, without altering its transcriptional patterns. RNA processing in the nucleus and the export of mRNA to the cytoplasm provide opportunities for gene regulation that are not available in prokaryotes.

RNA Processing In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Alternative RNA splicing significantly expands the repertoire of a set of genes. In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript. Alternative RNA splicing significantly expands the repertoire of a set of genes.

Exons DNA Troponin T gene Primary RNA transcript RNA splicing mRNA or
Fig Exons DNA Troponin T gene Primary RNA transcript Figure Alternative RNA splicing of the troponin T gene RNA splicing mRNA or

Eukaryotic mRNA is more long lived than prokaryotic mRNA
mRNA Degradation The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis Eukaryotic mRNA is more long lived than prokaryotic mRNA Life span is determined in part by sequences in the leader and trailer regions The shortening of the poly-A tail triggers the enzymatic removal of the 5 cap. Followed by degradation of the mRNA by nucleases. The life span of an mRNA molecule is an important factor in determining the pattern of protein synthesis. Prokaryotic mRNA molecules are typically degraded after only a few minutes. Eukaryotic mRNAs typically last for hours, days, or weeks. In red blood cells, mRNAs for hemoglobin polypeptides are unusually stable and are translated repeatedly. A common pathway of mRNA breakdown begins with enzymatic shortening of the poly-A tail. This triggers the enzymatic removal of the 5 cap. The next step is rapid degradation of the mRNA by nucleases. Nucleotide sequences in the untranslated trailer region (UTR) at the 3 end affect mRNA stability. Transferring such a sequence from a short-lived mRNA to a normally stable mRNA results in quick mRNA degradation.

Initiation of Translation
The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA Alternatively, translation of all mRNAs in a cell may be regulated simultaneously For example, translation initiation factors are simultaneously activated in an egg following fertilization The initiation of translation of an mRNA can be blocked by regulatory proteins that bind to specific sequences or structures within the 5 UTR of the mRNA, preventing ribosome attachment. The mRNAs present in the eggs of many organisms lack poly-A tails of sufficient length to allow initiation of translation. During embryonic development, a cytoplasmic enzyme adds more adenine nucleotides so that translation can begin at the appropriate time. Translation of all the mRNAs in a eukaryotic cell may be regulated simultaneously by the activation or inactivation of the protein factors required to initiate translation. This mechanism starts the translation of mRNAs that are stored in eggs. Just after fertilization, translation is triggered by the sudden activation of translation initiation factors, resulting in a burst of protein synthesis. Some plants and algae store mRNAs during periods of darkness. Light triggers the reactivation of the translational apparatus.

Protein Processing and Degradation
After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them Often, eukaryotic polypeptides are processed to yield functional proteins. For example, cleavage of pro-insulin forms the active hormone. Many proteins must undergo chemical modifications before they are functional. Phosphate groups activate regulatory proteins. Sugars are added to cell-surface proteins, which are then transported to their target destination in the cell. Regulation may occur at any of the steps involved in modifying or transporting a protein. Finally, the length of time a protein functions before it is degraded is strictly regulated. Proteins such as the cyclins that regulate the cell cycle must be relatively short-lived. To mark a protein for destruction, the cell attaches a small protein called ubiquitin to it. Animation: Protein Processing Animation: Protein Degradation

Giant protein complexes called proteasomes recognize and degrade the tagged proteins.
and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Figure Degradation of a protein by a proteasome Protein entering a proteasome Mutations making specific cell cycle proteins impervious to proteasome degradation can lead to cancer.

Only a small fraction of DNA codes for proteins, rRNA, and tRNA
Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression Only a small fraction of DNA codes for proteins, rRNA, and tRNA A significant amount of the genome may be transcribed into noncoding RNAs (non-protein-coding) Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration Only 1.5% of the human genome codes for proteins. Of the remainder, only a very small fraction consists of genes for ribosomal RNA and transfer RNA. Until recently, it was assumed that most of the rest of the DNA was untranscribed. Recent data have challenged that assumption, however. In one study of two human chromosomes, it was found that ten times as much of the genome was transcribed as was predicted by the number of protein-coding exons present. Introns accounted for only a small fraction of this transcribed, nontranslated RNA. A significant amount of the genome may be transcribed into non–protein-coding RNAs (or noncoding RNAs), including a variety of small RNAs. These discoveries suggest that there may be a large, diverse population of RNA molecules that play crucial roles in regulating gene expression in the cell.

Effects on mRNAs by MicroRNAs and Small Interfering RNAs
MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA These can degrade mRNA or block its translation In the past few years, researchers have found small, single-stranded RNA molecules called microRNAs (miRNAs) that bind to complementary sequences in mRNA molecules. miRNAs are formed from longer RNA precursors that fold back on themselves to form one or more short, double-stranded hairpin structures stabilized by hydrogen bonding. An enzyme called Dicer cuts each hairpin into a short, double-stranded fragment of about 20 nucleotide pairs. One of the two strands is degraded. The other strand (miRNA) associates with a protein complex and directs the complex to any mRNA molecules that have a complementary sequence. The miRNA–protein complex either degrades the target mRNA or blocks its translation. It is estimated that the expression of up to one-third of all human genes may be regulated by miRNAs.

(b) Generation and function of miRNAs
Fig Hairpin miRNA Hydrogen bond Dicer miRNA miRNA- protein complex 5 3 (a) Primary miRNA transcript Figure Regulation of gene expression by miRNAs mRNA degraded Translation blocked (b) Generation and function of miRNAs

RNAi is caused by small interfering RNAs (siRNAs)
The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) RNAi is caused by small interfering RNAs (siRNAs) siRNAs and miRNAs are similar but form from different RNA precursors The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi). RNA interference is due to small interfering RNAs (siRNAs), which are similar in size and function to miRNAs and are generated by similar mechanisms in eukaryotic cells. Both miRNAs and siRNAs can associate with the same proteins, with similar results. The distinction between these molecules is the nature of the precursor molecules from which they are formed. Each miRNA is formed from a single hairpin in the precursor RNA, whereas siRNAs are formed from longer, double-stranded RNA molecules that give rise to many siRNAs. Cellular RNAi pathways lead to the destruction of RNAs and may have originated as a natural defense against infection by double-stranded RNA viruses. The fact that the RNAi pathway can also affect the expression of nonviral cellular genes may reflect a different evolutionary origin for the RNAi pathway. Some species possess long, double-stranded RNAs that arise from cellular genes and are cut into small RNAs that block various steps in gene expression. Whatever their origin, RNAi plays an important role in regulating gene expression in the cell.

Chromatin Remodeling and Silencing of Transcription by Small RNAs
siRNAs play a role in heterochromatin formation and can block large regions of the chromosome Small RNAs may also block transcription of specific genes Small RNAs can cause remodeling of chromatin structure. In yeast, siRNAs are necessary for the formation of heterochromatin at the centromeres of chromosomes. Experimental evidence suggests that an RNA transcript produced from DNA in the centromeric region of the chromosome is copied into double-stranded RNA by a yeast enzyme and then processed into siRNAs. The siRNAs associate with a protein complex, targeting the complex back to the centromeric sequences of DNA. The proteins in the complex recruit enzymes to modify the chromatin, turning it into the highly condensed centromeric heterochromatin. When the enzyme Dicer is inactivated in chicken and mouse cells, heterochromatin fails to form at the centromeres. Related mechanisms may also block the transcription of specific genes. Many of the miRNAs that have been characterized play important roles in embryonic development, the ultimate example of an elaborate program of regulated gene expression.

Gene expression orchestrates the developmental programs of animals
Concept 18.4: A program of differential gene expression leads to the different cell types in a multicellular organism During embryonic development, a fertilized egg gives rise to many different cell types Cell types are organized successively into tissues, organs, organ systems, and the whole organism Gene expression orchestrates the developmental programs of animals In the development of most multicellular organisms, a single-celled zygote gives rise to cells of many different types. Each type has a different structure and corresponding function. Cells of different types are organized into tissues, tissues into organs, organs into organ systems, and organ systems into the whole organism. Thus, the process of embryonic development must give rise not only to cells of different types but also to higher-level structures arranged in a particular way in three dimensions.

A Genetic Program for Embryonic Development
The transformation from zygote to adult results from cell division, cell differentiation, and morphogenesis (a) Fertilized eggs of a frog (b) Newly hatched tadpole

Cell differentiation is the process by which cells become specialized in structure and function
The physical processes that give an organism its shape constitute morphogenesis (creation of form) Differential gene expression results from genes being regulated differently in each cell type Materials in the egg can set up gene regulation that is carried out as cells divide As a zygote develops into an adult organism, its transformation results from three interrelated processes: cell division, cell differentiation, and morphogenesis. Through a succession of mitotic cell divisions, the zygote gives rise to a large number of cells. Cell division alone would produce only a great ball of identical cells. During development, cells become specialized in structure and function, undergoing cell differentiation. Different kinds of cells are organized into tissues and organs. The physical processes that give an organism its shape constitute morphogenesis, the “creation of form.”

Cytoplasmic Determinants and Inductive Signals
An egg’s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg Cytoplasmic determinants are maternal substances in the egg that influence early development As the zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different gene expression Cell division, cell differentiation, and morphogenesis have their basis in cellular behavior. Morphogenesis can be traced back to changes in the shape and motility of cells in the various embryonic regions. The activities of a cell depend on the genes it expresses and the proteins it produces. Because almost all cells in an organism have the same genome, differential gene expression results from differential gene regulation in different cell types. Why are different sets of activators present in different cell types? One important source of information early in development is the egg’s cytoplasm, which contains both RNA and proteins encoded by the mother’s DNA. Cytoplasmic materials are distributed unevenly in the unfertilized egg. Maternal substances that influence the course of early development are called cytoplasmic determinants. These substances regulate the expression of genes that affect the developmental fate of the cell. After fertilization, the cell nuclei resulting from mitotic division of the zygote are exposed to different cytoplasmic environments. The set of cytoplasmic determinants a particular cell receives helps determine its developmental fate by regulating expression of the cell’s genes during cell differentiation.

(a) Cytoplasmic determinants in the egg
Fig a Unfertilized egg cell Sperm Nucleus Fertilization Two different cytoplasmic determinants Zygote Mitotic cell division Figure Sources of developmental information for the early embryo Two-celled embryo (a) Cytoplasmic determinants in the egg

The other important source of developmental information is the environment around the cell, especially signals from nearby embryonic cells In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells Thus, interactions between cells induce differentiation of specialized cell types The other important source of developmental information is the environment around the cell, especially signals impinging on an embryonic cell from other nearby embryonic cells. In animals, these signals include contact with cell-surface molecules on neighboring cells and the binding of growth factors secreted by neighboring cells. These signals cause changes in the target cells, a process called induction. The molecules conveying these signals within the target cells are cell-surface receptors and other proteins expressed by the embryo’s own genes. The signal molecules send a cell down a specific developmental path by causing a change in its gene expression that eventually results in observable cellular changes.

(b) Induction by nearby cells
Fig b NUCLEUS Early embryo (32 cells) Signal transduction pathway Signal receptor Figure Sources of developmental information for the early embryo Signal molecule (inducer) (b) Induction by nearby cells

Cellular Differentiation
During embryonic development, cells become visibly different in structure and function as they differentiate. The earliest changes that set a cell on a path to specialization show up only at the molecular level. Molecular changes in the embryo drive the process, called determination, that leads to the observable differentiation of a cell. Once it has undergone determination, an embryonic cell is irreversibly committed to its final fate. 61

Sequential Regulation of Gene Expression During Cellular Differentiation
The outcome of determination—observable cell differentiation—is caused by the expression of genes that encode tissue-specific proteins. These proteins give a cell its characteristic structure and function. Differentiation begins with the appearance of cell-specific mRNAs and is eventually observable in the microscope as changes in cellular structure.

In most cases, the pattern of gene expression in a differentiated cell is controlled at the level of transcription. Cells produce the proteins that allow them to carry out their specialized roles in the organism. For example, liver cells specialize in making albumin, while lens cells specialize in making crystalline. Similarly, skeletal muscle cells have high concentrations of proteins specific to muscle tissues, such as a muscle-specific version of the contractile proteins myosin and actin. In most cases, the pattern of gene expression in a differentiated cell is controlled at the level of transcription. Cells produce the proteins that allow them to carry out their specialized roles in the organism. For example, liver cells specialize in making albumin, while lens cells specialize in making crystalline. Similarly, skeletal muscle cells have high concentrations of proteins specific to muscle tissues, such as a muscle-specific version of the contractile proteins myosin and actin. Skeletal muscle cells also have membrane receptor proteins that detect signals from nerve cells.

Myoblasts produce muscle-specific proteins and form skeletal muscle cells
MyoD is one of several “master regulatory genes” that produce proteins that commit the cell to becoming skeletal muscle The MyoD protein is a transcription factor that binds to enhancers of various target genes Muscle cells develop from embryonic precursors that have the potential to develop into a number of alternative cell types, including cartilage cells, fat cells, or multinucleate muscle cells. Particular conditions commit certain embryonic precursors to become muscle cells. Although the committed cells are unchanged, they are now myoblasts. Eventually, the myoblasts begin to synthesize muscle-specific proteins and fuse to form mature, elongated, multinucleate skeletal muscle cells. Researchers have worked out the events at the molecular level that lead to muscle cell determination growing myoblasts in culture and analyzing them with molecular biology techniques. Researchers isolated different genes by causing each to be expressed in a separate embryonic precursor cell and then looking for differentiation into myoblasts and muscle cells. They identified several “master regulatory genes” that, when transcribed and translated, commit the cells to become skeletal muscle. One of these master regulatory genes is called myoD. myoD encodes MyoD protein, a transcription factor that binds to specific control elements in the enhancers of various target genes and stimulates their expression. Some target genes for MyoD encode for other muscle-specific transcription factors. MyoD also stimulates expression of the myoD gene itself, perpetuating its effect in maintaining the cell’s differentiated state. All the genes activated by MyoD have enhancer control elements recognized by MyoD and are thus coordinately controlled. The secondary transcription factors activate the genes for proteins such as myosin and actin to confer the unique properties of skeletal muscle cells. The MyoD protein is capable of changing fully differentiated non-muscle cells into muscle cells. Not all cells can be transformed by MyoD, however. Nontransforming cells may lack a combination of regulatory proteins in addition to MyoD.

(fully differentiated cell)
Fig Nucleus Master regulatory gene myoD Other muscle-specific genes DNA Embryonic precursor cell OFF OFF mRNA OFF MyoD protein (transcription factor) Myoblast (determined) Figure Determination and differentiation of muscle cells mRNA mRNA mRNA mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins MyoD Another transcription factor Part of a muscle fiber (fully differentiated cell)

Pattern Formation: Setting Up the Body Plan
Pattern formation is the development of a spatial organization of tissues and organs In animals, pattern formation begins with the establishment of the major axes Positional information, the molecular cues that control pattern formation, tells a cell its location relative to the body axes and to neighboring cells Cytoplasmic determinants and inductive signals contribute to pattern formation, the development of spatial organization in which the tissues and organs of an organism are all in their characteristic places. Pattern formation begins in the early embryo, when the major axes of an animal are established. Before specialized tissues and organs form, the relative positions of a bilaterally symmetrical animal’s head and tail, right and left sides, and back and front are established. These are the three major body axes of the animal. The molecular cues that control pattern formation, positional information, are provided by cytoplasmic determinants and inductive signals. These signals tell a cell its location relative to the body axes and to neighboring cells. The signals also determine how the cell and its progeny will respond to future molecular signals.

Pattern formation has been extensively studied in the fruit fly Drosophila melanogaster
Combining anatomical, genetic, and biochemical approaches, researchers have discovered developmental principles common to many other species, including humans Pattern formation has been most extensively studied in Drosophila melanogaster, where genetic approaches have had spectacular success. These studies have established that genes control development, and they have identified the key roles that specific molecules play in defining position and directing differentiation. Combining anatomical, genetic, and biochemical approaches in the study of Drosophila development, researchers have discovered developmental principles common to many other species, including humans.

The Life Cycle of Drosophila
In Drosophila, cytoplasmic determinants in the unfertilized egg determine the axes before fertilization After fertilization, the embryo develops into a segmented larva with three larval stages

Head Thorax Abdomen 0.5 mm Dorsal Right BODY AXES Anterior Posterior
Fig a Head Thorax Abdomen 0.5 mm Dorsal Right BODY AXES Anterior Posterior Figure Key developmental events in the life cycle of Drosophila Fruit flies and other arthropods have a modular construction, an ordered series of segments. These segments make up the three major body parts: the head, thorax (with wings and legs), and abdomen. Like other bilaterally symmetrical animals, Drosophila has an anterior-posterior axis, a dorsal-ventral axis, and a right-left axis. Cytoplasmic determinants in the unfertilized egg provide positional information for two developmental axes (anterior-posterior and dorsal-ventral axis) before fertilization. Left Ventral (a) Adult

(b) Development from egg to larva
Fig b Follicle cell 1 Egg cell developing within ovarian follicle Nucleus Egg cell Nurse cell 2 Unfertilized egg Egg shell Depleted nurse cells Fertilization Laying of egg 3 Fertilized egg Embryonic development Figure Key developmental events in the life cycle of Drosophila The Drosophila egg develops in the female’s ovary, surrounded by ovarian cells called nurse cells and follicle cells that supply the egg cell with nutrients, mRNAs, and other substances needed for development. Development of the fruit fly from egg cell to adult fly occurs in a series of discrete stages. The egg forms a segmented larva, which goes through three larval stages. The fly larva forms a pupal cocoon within which it metamorphoses into an adult fly. 4 Segmented embryo 0.1 mm Body segments Hatching 5 Larval stage (b) Development from egg to larva

Genetic Analysis of Early Development: Scientific Inquiry
Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric Wieschaus won a Nobel 1995 Prize for decoding pattern formation in Drosophila Lewis demonstrated that homeotic genes direct the developmental process In the 1940s, Edward B. Lewis demonstrated that the study of mutants can be used to investigate Drosophila development. Lewis studied bizarre developmental mutations and located the mutations on the fly’s genetic map. This research provided the first concrete evidence that genes somehow direct the developmental process.

Eye Leg Antenna Wild type Mutant Fig. 18-18
Figure Abnormal pattern formation in Drosophila Wild type Mutant

They found 120 genes essential for normal segmentation
Nüsslein-Volhard & Wieschaus decided to study all the genes segment formation in Drosophilia They created mutants, conducted breeding experiments, and looked for corresponding genes Breeding experiments were complicated by embryonic lethals, embryos with lethal mutations They found 120 genes essential for normal segmentation In 1995, Nüsslein-Volhard, Wieschaus, and Lewis were awarded the Nobel Prize. In the late 1970s, Christiane Nüsslein-Volhard and Eric Weischaus pushed the understanding of early pattern formation to the molecular level. Their goal was to identify all the genes that affect segmentation in Drosophila, but they faced three problems. First, because Drosophila has about 13,700 genes, there could be either only a few genes affecting segmentation or so many that the pattern would be impossible to discern. Second, mutations that affect segmentation are likely to be embryonic lethals, leading to death at the embryonic or larval stage. Because flies with embryonic lethal mutations never reproduce, they cannot be bred for study. Nüsslein-Volhard and Wieschaus focused on recessive mutations that could be propagated in heterozygous flies. Third, because of maternal effects on axis formation in the egg, the researchers also needed to study maternal genes. After exposing flies to mutagenic chemicals, Nüsslein-Volhard and Wieschaus looked for dead embryos and larvae with abnormal segmentation (such as two heads or two tails) among the fly’s descendents. Through appropriate crosses, they could identify living heterozygotes carrying embryonic lethal mutations. They hoped that the segmental abnormalities would suggest how the affected genes normally functioned. Nüsslein-Volhard and Wieschaus identified 1,200 genes essential for embryonic development. About 120 of these were essential for pattern formation leading to normal segmentation. After several years, they were able to group the genes by general function, map them, and clone many of them. Their results, combined with Lewis’s early work, created a coherent picture of Drosophila development. In 1995, Nüsslein-Volhard, Wieschaus, and Lewis were awarded the Nobel Prize.

Animation: Development of Head-Tail Axis in Fruit Flies
Axis Establishment Maternal effect genes encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila These maternal effect genes are also called egg-polarity genes because they control orientation of the egg and consequently the fly One group of genes sets up the anterior-posterior axis, while a second group establishes the dorsal-ventral axis. Cytoplasmic determinants establish the axes of the Drosophila body. Substances are produced under the direction of maternal effect genes that are deposited in the unfertilized egg. A maternal effect gene is a gene that, when mutant in the mother, results in a mutant phenotype in the offspring, regardless of the offspring’s own genotype. In fruit fly development, maternal effect genes encode proteins or mRNA that are placed in the egg while it is still in the ovary. When the mother has a mutation in a maternal effect gene, she makes a defective gene product (or none at all) and her eggs will not develop properly when fertilized. Maternal effect genes are also called egg-polarity genes because they control the orientation of the egg and consequently the fly. One group of genes sets up the anterior-posterior axis, while a second group establishes the dorsal-ventral axis. Animation: Development of Head-Tail Axis in Fruit Flies

Bicoid: A Morphogen Determining Head Structures
One maternal effect gene, the bicoid gene, affects the front half of the body An embryo whose mother has a mutant bicoid gene lacks the front half of its body and has duplicate posterior structures at both ends One gene called bicoid affects the front half of the body. An embryo whose mother has a mutant bicoid gene lacks the front half of its body and has duplicate posterior structures at both ends. This suggests that the product of the mother’s bicoid gene is essential for setting up the anterior end of the fly. It also suggests that the gene’s products are concentrated at the future anterior end.

EXPERIMENT Tail Head Wild-type larva Tail Tail Mutant larva (bicoid)
Fig a EXPERIMENT Tail Head A8 T1 T2 T3 A7 A1 A6 A2 A3 A4 A5 Wild-type larva Tail Tail Figure Is Bicoid a morphogen that determines the anterior end of a fruit fly? A8 A8 A7 A7 A6 Mutant larva (bicoid)

RESULTS Anterior end Bicoid mRNA in mature unfertilized egg
Fig b RESULTS Fertilization, translation of bicoid mRNA 100 µm Anterior end Figure Is Bicoid a morphogen that determines the anterior end of a fruit fly? Bicoid mRNA in mature unfertilized egg Bicoid protein in early embryo

CONCLUSION Nurse cells Egg bicoid mRNA Developing egg
Fig c CONCLUSION Nurse cells Egg bicoid mRNA Developing egg Figure Is Bicoid a morphogen that determines the anterior end of a fruit fly? Bicoid mRNA in mature unfertilized egg Bicoid protein in early embryo

This phenotype suggests that the product of the mother’s bicoid gene is concentrated at the future anterior end This hypothesis is an example of the gradient hypothesis, in which gradients of substances called morphogens establish an embryo’s axes and other features This is a specific version of a general gradient hypothesis, in which gradients of morphogens establish an embryo’s axes and other features. Using DNA technology and biochemical methods, researchers were able to clone the bicoid gene and use it as a probe for bicoid mRNA in the egg. As predicted, the bicoid mRNA is concentrated at the extreme anterior end of the egg cell. Bicoid mRNA is produced in nurse cells, transferred to the egg via cytoplasmic bridges, and anchored to the cytoskeleton at the anterior end of the egg. After the egg is fertilized, bicoid mRNA is transcribed into Bicoid protein, which diffuses from the anterior end toward the posterior, resulting in a gradient of proteins in the early embryo. Injections of pure bicoid mRNA into various regions of early embryos resulted in the formation of anterior structures at the injection sites as the mRNA was translated into protein.

The bicoid research is important for three reasons:
– It identified a specific protein required for some early steps in pattern formation – It increased understanding of the mother’s role in embryo development – It demonstrated a key developmental principle that a gradient of molecules can determine polarity and position in the embryo The bicoid research is important for three reasons. It identified a specific protein required for some of the earliest steps in pattern formation. It increased our understanding of the mother’s role in the development of an embryo. As one developmental biologist put it, “Mom tells Junior which way is up.” It demonstrated a key developmental principle: that a gradient of molecules can determine polarity and position in the embryo. Gradients of specific proteins determine the posterior end as well as the anterior end and also are responsible for establishing the dorsal-ventral axis. Later, positional information operating at a finer scale establishes a specific number of correctly oriented segments and triggers the formation of each segment’s characteristic structures.

Concept 18.5: Cancer results from genetic changes that affect cell cycle control
Gene regulation systems that go wrong during cancer are the same systems that play important roles in embryonic development, the immune response & other biological processes Genes that normally regulate cell growth & division during the cell cycle include genes for growth factors, their receptors & the intracellular molecules of signaling pathways Mutations altering any of these genes in somatic cells can lead to cancer Cancer is a set of diseases in which cells escape the control mechanisms that normally regulate cell growth and division. The gene regulation systems that go wrong during cancer are the very same systems that play important roles in embryonic development, the immune response, and other biological processes. The genes that normally regulate cell growth and division during the cell cycle include genes for growth factors, their receptors, and the intracellular molecules of signaling pathways. Mutations altering any of these genes in somatic cells can lead to cancer.

Types of Genes Associated with Cancer
The agent of such changes can be random spontaneous mutations or environmental influences such as chemical carcinogens, X-rays, or certain viruses. Since then, scientists have recognized a number of tumor viruses that cause cancer in various animals, including humans. Viruses play a role in about 15% of human cancers. The agent of such changes can be random spontaneous mutations or environmental influences such as chemical carcinogens, X-rays, or certain viruses. In 1911, Peyton Rous discovered a virus that causes cancer in chickens. Since then, scientists have recognized a number of tumor viruses that cause cancer in various animals, including humans. The Epstein-Barr virus, which causes infectious mononucleosis, has been linked to several types of cancer, including Burkitt’s lymphoma. Papilloma viruses are associated with cervical cancer, while the HTLV-1 virus causes a form of adult leukemia. Viruses play a role in about 15% of human cancers.

Oncogenes and Proto-Oncogenes
Oncogenes are cancer-causing genes Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle Cancer-causing genes, oncogenes, were initially discovered in retroviruses, but close counterparts, proto-oncogenes, have been found in the genomes of humans and other animals. The products of proto-oncogenes are proteins that stimulate normal cell growth and division and play essential functions in normal cells.

within a control element within the gene
Fig Proto-oncogene DNA Translocation or transposition: Gene amplification: Point mutation: within a control element within the gene New promoter Oncogene Oncogene Figure Genetic changes that can turn proto-oncogenes into oncogenes A proto-oncogene becomes an oncogene following genetic changes that lead to an increase in the proto-oncogene’s protein production or in the intrinsic activity of each protein molecule. These genetic changes include movement of DNA within the genome, amplification of the proto-oncogene, and point mutations in a control element or the proto-oncogene itself. Cancer cells frequently have chromosomes that have been broken and rejoined incorrectly. A fragment may be moved to a location near an active promoter or other control element. Movement of transposable elements may also place a more active promoter near a proto-oncogene, increasing its expression. Amplification increases the number of copies of the proto-oncogene in the cell. A point mutation in the promoter or enhancer of a proto-oncogene may increase its expression. A point mutation in the coding sequence may lead to translation of a protein that is more active or longer-lived. All of these mechanisms can lead to abnormal stimulation of the cell cycle, putting the cell on the path to malignancy. Normal growth- stimulating protein in excess Normal growth-stimulating protein in excess Normal growth- stimulating protein in excess Hyperactive or degradation- resistant protein

Proto-oncogenes can be converted to oncogenes by
Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase Amplification of a proto-oncogene: increases the number of copies of the gene Point mutations in the proto-oncogene or its control elements: causes an increase in gene expression

Tumor-Suppressor Genes
Tumor-suppressor genes help prevent uncontrolled cell growth Mutations that decrease normal activity of tumor-suppressor proteins may contribute to cancer onset Tumor-suppressor proteins Repair damaged DNA Control cell adhesion Inhibit the cell cycle in the cell-signaling pathway The normal products of tumor-suppressor genes inhibit cell division. Any decrease in the normal activity of a tumor-suppressor protein may contribute to cancer. Some tumor-suppressor proteins normally repair damaged DNA, preventing the accumulation of cancer-causing mutations. Other tumor-suppressor proteins control the adhesion of cells to each other or to an extracellular matrix, which is crucial for normal tissues and often absent in cancers. Still others are components of cell-signaling pathways that inhibit the cell cycle.

Interference with Normal Cell-Signaling Pathways
Mutations in the ras proto-oncogene and p53 tumor-suppressor gene occur in 30% and 50% of human cancers, respectively The Ras protein, the product of the ras gene, is a G protein that relays a growth signal from a growth factor receptor on the plasma membrane to a cascade of protein kinases. Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division The proteins encoded by many proto-oncogenes and tumor-suppressor genes are components of cell-signaling pathways. Mutations in the products of two key genes, the ras proto-oncogene and the p53 tumor-suppressor gene, occur in 30% and 50% of human cancers, respectively. Both the Ras protein and the p53 protein are components of signal-transduction pathways that convey external signals to the DNA in the cell’s nucleus. The Ras protein, the product of the ras gene, is a G protein that relays a growth signal from a growth factor receptor on the plasma membrane to a cascade of protein kinases. At the end of the pathway is the synthesis of a protein that stimulates the cell cycle. Many ras oncogenes have a point mutation that leads to a hyperactive version of the Ras protein that can issue signals on its own, resulting in excessive cell division in the absence of the appropriate growth factor.

(a) Cell cycle–stimulating pathway
Fig a 1 Growth factor 1 MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own Ras 3 G protein GTP Ras GTP 2 Receptor 4 Protein kinases (phosphorylation cascade) NUCLEUS 5 Transcription factor (activator) DNA Figure Signaling pathways that regulate cell division Gene expression Protein that stimulates the cell cycle (a) Cell cycle–stimulating pathway

The p53 Gene – a tumor suppressor
The p53 protein is a specific transcription factor for the synthesis of several cell cycle-inhibiting proteins. Damage to the cell’s DNA acts as a signal that leads to expression of the p53 gene. The p53 protein can activate the p21 gene, whose product halts the cell cycle by binding to cyclin-dependent kinases, allowing time for DNA repair. The p53 protein can also turn on genes directly involved in DNA repair. When DNA damage is irreparable, the p53 protein can activate apoptosis. The p53 gene, named for its 53,000-dalton protein product, is a tumor-suppressor gene. The p53 protein is a specific transcription factor for the synthesis of several cell cycle-inhibiting proteins. The p53 gene has been called the “guardian angel of the genome.” Damage to the cell’s DNA acts as a signal that leads to expression of the p53 gene. The p53 protein can activate the p21 gene, whose product halts the cell cycle by binding to cyclin-dependent kinases, allowing time for DNA repair. The p53 protein can also turn on genes directly involved in DNA repair. When DNA damage is irreparable, the p53 protein can activate “suicide genes” whose protein products cause cell death by apoptosis. A mutation that knocks out the p53 gene can lead to excessive cell growth and cancer.

(b) Cell cycle–inhibiting pathway
Fig b 2 Protein kinases MUTATION Defective or missing transcription factor, such as p53, cannot activate 3 Active form of p53 UV light 1 DNA damage in genome DNA Figure Signaling pathways that regulate cell division Protein that inhibits the cell cycle (b) Cell cycle–inhibiting pathway

(c) Effects of mutations
Fig c EFFECTS OF MUTATIONS Protein overexpressed Protein absent Cell cycle overstimulated Increased cell division Cell cycle not inhibited (c) Effects of mutations Figure Signaling pathways that regulate cell division

The Multistep Model of Cancer Development
Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes More than one somatic mutation is generally needed to produce the changes characteristic of a full-fledged cancer cell. If cancer results from an accumulation of mutations, and if mutations occur throughout life, then the longer we live, the more likely we are to develop cancer. Colorectal cancer, with 135,000 new cases and 60,000 deaths in the United States each year, illustrates a multistep cancer path. The first sign is often a polyp, a small benign growth in the colon lining. The cells of the polyp look normal but divide unusually frequently. Through gradual accumulation of mutations that activate oncogenes and knock out tumor-suppressor genes, the polyp can develop into a malignant tumor. A ras oncogene and a mutated p53 tumor-suppressor gene are usually involved. About a half dozen DNA changes must occur for a cell to become fully cancerous. These changes usually include the appearance of at least one active oncogene and the mutation or loss of several tumor-suppressor genes. Because mutant tumor-suppressor alleles are usually recessive, mutations must knock out both alleles. Most oncogenes behave like dominant alleles and require only one mutation. In many malignant tumors, the gene for telomerase is activated, removing a natural limit on the number of times the cell can divide.

DCC – deleted in colorectal cancer
Fig About a half dozen DNA changes must occur for a cell to become fully cancerous. In many malignant tumors, the gene for telomerase is activated, removing a natural limit on the number of times the cell can divide. Colon EFFECTS OF MUTATIONS Loss of tumor- suppressor gene APC (or other) 1 Activation of ras oncogene 2 Loss of tumor-suppressor gene p53 4 Colon wall Loss of tumor-suppressor gene DCC 3 Additional mutations 5 Figure A multistep model for the development of colorectal cancer Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Malignant tumor (carcinoma) DCC – deleted in colorectal cancer

Inherited Predisposition and Other Factors Contributing to Cancer
Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli are common in individuals with colorectal cancer Mutations in the BRCA1 or BRCA2 gene are found in at least half of inherited breast cancers which account for only 5% and 10% of breast cancer cases The fact that multiple genetic changes are required to produce a cancer cell helps explain the predispositions to cancer that run in families. An individual inheriting an oncogene or a mutant allele of a tumor-suppressor gene is one step closer to accumulating the necessary mutations for cancer to develop. Geneticists are devoting much effort to finding inherited cancer alleles so that a predisposition to certain cancers can be detected early in life. About 15% of colorectal cancers involve inherited mutations. Many of these mutations affect the tumor-suppressor gene adenomatous polyposis coli, or APC. Normal functions of the APC gene include regulation of cell migration and adhesion. Even in patients with no family history of the disease, APC is mutated in about 60% of colorectal cancers. Between 5% and 10% of breast cancer cases show an inherited predisposition. Breast cancer is the second most common type of cancer in the United States, annually striking more than 180,000 women and leading to 40,000 deaths. Mutations in one of two tumor-suppressor genes, BRCA1 and BRCA2, increase the risk of breast and ovarian cancer and are found in at least half of inherited breast cancers. BRCA stands for breast cancer. A woman who inherits one mutant BRCA1 allele has a 60% probability of developing breast cancer before age 50 (versus a 2% probability in an individual with two normal alleles). BRCA1 and BRCA2 are considered tumor-suppressor genes because their wild-type alleles protect against breast cancer and because their mutant alleles are recessive. Recent evidence suggests that the BRCA2 protein is directly involved in repairing breaks that occur in both strands of DNA. Because DNA breakage can contribute to cancer, the risk of cancer can be lowered by minimizing exposure to DNA-damaging agents, such as ultraviolet radiation in sunlight and the chemicals found in cigarette smoke. Viruses can contribute to the development of cancer by integrating their genetic material into the DNA of infected cells. Viral integration may donate an oncogene to a cell, disrupt a tumor-suppressor gene, or convert a proto-oncogene to an oncogene. Some viruses produce proteins that inactivate p53 and other tumor-suppressor proteins, making the cell more prone to becoming cancerous.

Mary-Claire King – UW professor
Human geneticist She is known for 3 major accomplishments: identifying breast cancer genes demonstrating that humans & chimpanzees are 99% genetically identical applying genomic sequencing to identify victims of human rights abuses. Bachelor’s in mathematics Ph.D. in genetics and epidemiology (wiki)

Figure 18.23 Tracking the molecular basis of breast cancer

Protein processing and degradation
Fig. 18-UN4 Chromatin modification Transcription • Genes in highly compacted chromatin are generally not transcribed. • Regulation of transcription initiation: DNA control elements bind specific transcription factors. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. • DNA methylation generally reduces transcription. • Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNA transcript mRNA degradation Translation mRNA or Fig. 18-UN4 Protein processing and degradation Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation.

Chromatin modification
Fig. 18-UN5 Chromatin modification • Small RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Chromatin modification Transcription Translation RNA processing • miRNA or siRNA can block the translation of specific mRNAs. mRNA degradation Translation Protein processing and degradation Fig. 18-UN5 mRNA degradation • miRNA or siRNA can target specific mRNAs for destruction.

You should now be able to:
Explain the concept of an operon and the function of the operator, repressor, and corepressor Explain the adaptive advantage of grouping bacterial genes into an operon Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control

Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription Define control elements and explain how they influence transcription Explain the role of promoters, enhancers, activators, and repressors in transcription control

Explain how eukaryotic genes can be coordinately expressed
Describe the roles played by small RNAs on gene expression Explain why determination precedes differentiation Describe two sources of information that instruct a cell to express genes at the appropriate time

Explain how maternal effect genes affect polarity and development in Drosophila embryos
Explain how mutations in tumor-suppressor genes can contribute to cancer Describe the effects of mutations to the p53 and ras genes

Regulation of Gene Expression

Similar presentations

Presentation on theme: "Regulation of Gene Expression"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Regulation of Gene Expression

Similar presentations

Presentation on theme: "Regulation of Gene Expression"— Presentation transcript:

Similar presentations

About project

Feedback