Chapter 18: Regulation of Gene Expression

Transcription

1 Chapter 18: Regulation of Gene Expression AP Biology

2 Overview: Conducting the Genetic Orchestra Prokaryotes and eukaryotes alter gene expression in response to changes in environmental conditions Multicellular eukaryotes must also develop and maintain multiple cell types Though multicellular eukaryotes have different types of cells, all of these cells contain the same genome A significant challenge in the gene regulation of these organisms is controlling the expression of different subsets of genes to create different cell types Gene expression is often regulated at the stage of transcription, but control at other levels of gene expression is also important RNA molecules play many roles in regulating gene expression in eukaryotes

3 Concept 18.1: Bacteria often respond to environmental change by regulating transcription

4 Regulation of Enzyme Activity and Production Natural selection has favored bacteria that produce only the products needed by that cell By doing so, these bacteria can conserve resources and energy for other important tasks Metabolic control occurs on 2 levels: First, cells can adjust the activity of enzymes that are already present by Fig feedback inhibition In this type of inhibition, the activity of an enzyme is inhibited by a product in an anabolic pathway Second, cells can adjust the production level of certain enzymes by regulating the expression of the genes encoding these enzymes Gene expression in bacteria is controlled by the operon model Feedback inhibition Tryptophan Precursor Enzyme 1 Enzyme 2 Enzyme 3 (a) Regulation of enzyme activity trpe gene trpd gene trpc gene trpb gene trpa gene Regulation of gene expression (b) Regulation of enzyme production

5 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 The operator controls access of RNA polymerase to the genes An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

6 Repressors The operon can be switched off by a protein repressor The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase (no transcription) A repressor protein is specific for the operator of a particular operon The repressor is the product of a separate regulatory gene Regulatory genes are expressed continuously, although at a low rate, so that a few repressor molecules are always present within the cell

7 Repressors (Continued) The binding of repressors to operators is reversible Operators can be in one of 2 states at any given time: One with repressor bound ( off mode) One without the repressor bound ( on mode) The relative duration of each state depends on the number of active repressor molecules present The repressor can be in an active or inactive form, depending on the presence of other molecules In its inactive form, the repressor has little affinity for its operator In its active form, a specific substrate binds to the repressor at an allosteric site, triggering a change in conformation These types of substrates are examples of molecules called corepressors that cooperates with a repressor protein to switch an operon off Ex) E. coli can synthesize the amino acid tryptophan

8 Operon On By default the trp operon is on and the genes for tryptophan synthesis are transcribed Fig. 18-3a Occurs when tryptophan is absent Repressor is inactive trp operon Promoter DNA trpr trpe trpd trpc trpb Regulatory Operator gene Start codon Stop codon 3 mrna RNA mrna 5 5 polymerase E D C B Protein Inactive repressor Promoter (a) Tryptophan absent, repressor inactive, operon on Genes of operon trpa Polypeptide subunits that make up enzymes for tryptophan synthesis A

9 Operon Off 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 Fig. 18-3b-2 Thus the trp operon is turned off (repressed) if tryptophan levels are high DNA No RNA made mrna Protein Tryptophan (corepressor) Active repressor (b) Tryptophan present, repressor active, operon off

10 Repressible and Inducible Operons: Two Types of Negative Gene Regulation There are 2 types of negative gene regulation: 1) A repressible operon is one that is usually on Binding of a repressor to the operator shuts off transcription Ex) trp operon 2) An inducible operon is one that is usually off A molecule called an inducer inactivates the repressor and turns on transcription Ex) lac operon

11 The lac Operon: An Inducible Operon The lac operon is an inducible operon found in E.coli cells This operon contains genes that code for enzymes used in the hydrolysis and metabolism of lactose Lactose metabolism begins with the hydrolysis of lactose into its component monosaccharides glucose and galactose This reaction is catalyzed by the enzyme β- galactosidase The gene for β-galactosidase is one of the 3 genes that code for enzymes that function in lactose utilization The entire transcription unit is under the command of a single operator and promoter A regulatory gene located outside the operon called laci codes for an allosteric repressor protein that can switch the operon off by binding to the operator

12 The lac Operon: Allolactose as an Inducer A molecule called an inducer inactivates the repressor to turn the lac operon on For the lac operon, the inducer is an isomer of lactose called allolactose Allolactose is formed in small amounts from lactose that enters the cell Allolactose binds to the lac repressor and alters its shape, preventing the repressor from binding to the operator Fig. 18-4b Without a bound repressor, the lac operon is transcribed into mrna, and the proteins needed for lactose utilization are produced lac operon DNA laci lacz lacy laca mrna 5 3 RNA polymerase mrna 5 Protein -Galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on

13 The lac Operon: Lactose Absent By itself, the lac repressor is active and switches the lac operon off Occurs due to the absence of lactose (and hence allolactose) Fig. 18-4a Regulatory gene Promoter Operator DNA laci lacz mrna 5 3 RNA polymerase No RNA made Protein Active repressor (a) Lactose absent, repressor active, operon off

14 Inducible vs. Repressible Enzymes The enzymes of the lactose pathway are referred to as inducible enzymes because their synthesis is induced by a chemical signal (allolactose) Inducible enzymes usually function in catabolic pathways The enzymes for tryptophan synthesis are referred to as repressible enzymes because their synthesis is repressed by high levels of the end product Repressible enzymes usually function in anabolic pathways Regulation of both the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor Gene regulation is said to be positive only when a regulatory protein interacts directly with the genome to switch transcription on

15 Positive Gene Regulation An example of positive gene regulation also involves the lac operon When glucose and lactose are both present, E.coli preferentially use glucose, since the enzymes for glycolysis are always present E.coli use lactose as an energy source only when glucose is in short supply When glucose is scarce, a small organic molecule called cyclic AMP (camp) accumulates In this case, the lac operon is subject to positive control through a stimulatory protein called catabolite activator protein (CAP), an activator of transcription CAP is activated by binding with camp, which allows it to attach to a specific site at the upstream end of the lac promoter This attachment increases the affinity of RNA polymerase for the promoter, thus accelerating transcription

16 CAP also helps regulate other operons that encode enzymes used in catabolic pathways Dual Control of the lac Operon When glucose levels in the cell increase, camp concentration decreases Without camp, CAP detaches from the lac operon Because CAP is inactive, the affinity of RNA polymerase for the promoter of the lac operon is lowered Transcription of the lac operon will thus proceed only at a low level, even in the presence of lactose Therefore, the lac operon is under dual control: Negative control by the lac repressor (like on-off switch) The state of the lac repressor (with or without bound allolactose) determines whether transcription of the lac operon s genes will occur at all Positive control by CAP (like volume control) The state of CAP (with or without bound camp) controls the rate of transcription if the operon is repressor-free

17 Concept 18.2: Eukaryotic gene expression can be regulated at any stage

18 Gene Expression and Cell Specialization All organisms must regulate which genes are expressed at any given time In multicellular organisms gene expression is essential for cell specialization To perform its role, each cell type must maintain a specific program of gene expression in which certain genes are expressed and others are not

19 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 A typical human cell expresses only ~20% of its genes at any given time Errors in gene expression can lead to diseases including cancer Gene expression in eukaryotic cells is regulated at many stages Each stage is a potential control point at which gene expression can be turned on or off, accelerated, or slowed down

20 Regulation of Gene Expression at Transcription In all organisms, a common control point for gene expression is at transcription Regulation at this stage is often in response to signals (hormones, signaling molecules) coming from outside the cell For this reason, the term gene expression is often equated with transcription for both bacteria and eukaryotes The greater complexity of eukaryotes, however, also provides opportunities for regulating gene expression at many additional stages

21 Fig Signal In this diagram, the colored boxes indicate processes most often regulated NUCLEUS Chromatin Chromatin modification Each color indicates the type of molecule affected (blue=dna, orange=rna, purple=protein) The nuclear envelope separating transcription and translation in eukaryotic cells offers opportunities for post-transcriptional control in the form of RNA processing DNA Cap Degradation of mrna RNA Gene Exon Intron Tail Gene available for transcription Transcription Primary transcript RNA processing mrna in nucleus Transport to cytoplasm mrna in cytoplasm Translatio n Polypeptide Protein processing CYTOPLASM In addition, eukaryotes have a greater variety of control mechanisms operating before transcription and after translation Degradation of protein Active protein Transport to cellular destination Cellular function

22 Regulation of Chromatin Structure The structural organization of chromatin not only packs a cell s DNA into a compact form that fits inside the nucleus, but it also helps regulate gene expression in several ways The location of a gene s promoter can affect whether a gene will be transcribed In addition, genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin also influence both chromatin structure and gene expression

23 Histone Modifications There is mounting evidence that chemical modifications to histones play a direct role in regulation of gene transcription The N-terminus of each histone molecule protrudes outward from the nucleosome Fig These histone tails are accessible to various modifying enzymes that catalyze the addition or removal of specific chemical groups DNA double helix Histone tails Amino acids available for chemical modification (a) Histone tails protrude outward from a nucleosome

24 Histone Acetylation DNA double helix Histone tails In histone acetylation, acetyl groups (-COCH3) are attached to positively charged lysines in histone tails Amino acids available for chemical modification When lysines are acetylated, their positive charges are neutralized As a result, histone tails no longer bind to neighboring nucleosomes (a) Histone tails protrude outward from a nucleosome This process loosens chromatin structure and allows transcription proteins easier access to genes, thereby promoting the initiation of transcription Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

25 Other Histone Modifications Several other chemical groups can be reversibly attached to amino acids in histone tails, including methyl and phosphate groups The addition of methyl groups (methylation) can condense chromatin The addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin The discovery that these and other 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, help determine chromatin configuration Chromatin configuration, in turn, has a direct influence on transcription

26 DNA Methylation Some enzymes can methylate certain bases of DNA itself DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species Ex) The inactivated mammalian X chromosome is generally more methylated than DNA that is actively transcribed DNA methylation can also cause long-term inactivation of genes in cellular differentiation Methylation patterns are passed on to successive generations of cells, so that cells keep a chemical record of what occurred during embryonic development A methylation pattern maintained in this way accounts for genomic imprinting in mammals In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

27 Epigenetic Inheritance Although chromatin modifications 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 Epigenetic variations might help explain why one identical twin acquires a genetically based disease (schizophrenia), but the other does not, despite their identical genomes

28 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 Once the chromatin of a gene is optimally modified for expression, the initiation of transcription is the next major step at which gene expression is regulated Involves proteins that bind to DNA and either facilitate or inhibit binding of RNA polymerase (transcription factors) Before looking at how eukaryotic cells control transcription, however, it is helpful to review the structure of a typical eukaryotic gene

29 In a typical eukaryotic gene, a cluster of proteins called a transcription initiation complex assembles on the promoter sequence at the upstream end of a gene One of these proteins (RNA polymerase II) then proceeds to transcribe the gene, producing a primary RNA transcript RNA processing follows, including enzymatic addition of a 5 cap and a poly-a tail, as well as splicing out of introns Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Fig Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types Enhancer (distal control elements) DNA Upstream Proximal control elements Promoter Exon Intron Exon Poly-A signal sequence Termination region Intron Exon Transcription Downstream Primary RNA transcript 5 Exon Intron Exon Intron Exon RNA processing Cleaved 3 end of primary transcript mrna Intron RNA 5 Cap 5 UTR Coding segment Start codon Poly-A signal Stop codon 3 UTR Poly-A 3

30 factors 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 proteincoding genes Most of these transcription factors do not bind DNA directly, but bind to proteins (including each other) and RNA polymerase II These protein-protein interactions are crucial to the initiation of eukaryotic transcription The interactions of general transcription factors and RNA polymerase II with a promoter, however, usually only lead to a low rate of transcription In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with another set of proteins called specific transcription

31 Proximal vs. Distal Control Elements Some of these specific transcription factors are called proximal control elements because they are located close to the promoter More distant groups of specific transcription factors called enhancers may be located 1000s of nucleotides upstream or downstream of a gene, or even within an intron These enhancers are referred to as distal control elements A given gene may have multiple enhancers, each active at a different time or in a different cell type or location within an organism Each enhancer, however, is only associated with one specific gene

32 Activators and Mediator Proteins In eukaryotes, the rate of gene expression can be strongly controlled by the binding of special proteins to the control elements of enhancers An activator is a protein that binds to an enhancer and stimulates transcription of a gene Protein-mediated bending of DNA is thought to bring bound activators in contact with another group of proteins called mediator proteins These mediator proteins will, in turn, interact with proteins at the promoter These multiple protein-protein interactions help assemble and position the initiation complex on the promoter Animation: Initiation of Transcription

33 Step 1: Activator proteins bind to distal control elements grouped as an enhancer in the DNA This particular enhancer has 3 binding sites Step 2: A DNA-bending protein brings the bound activator closer to the promoter General transcription factors, mediator proteins, and RNA polymerase are nearby Fig DNA Enhancer Activators Distal control element Promoter TATA box General transcription factors Gene Step 3: The activator bind to certain mediator proteins DNA-bending protein Group of mediator proteins and general transcription factors, helping them form RNA polymerase II an active transcription initiation complex on the promoter RNA polymerase II Transcription initiation complex RNA synthesis

34 Some transcription factors function as repressors, inhibiting expression of a particular gene Some repressors bind directly to control element DNA, like enhancers This may block activator binding or turn off transcription even when activators are bound Other repressors block the binding of activators to proteins that allow activators to bind to DNA Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription Activators may recruit proteins that acetylate histones near the promoters of specific genes, thereby promoting transcription Some repressors recruit proteins that deacetylate histones, leading to reduced transcription

35 Coordinately Controlled Genes in Eukaryotes Every gene whose transcription is stimulated by a particular chemical signal has a control element recognized by the same complex, regardless of its chromosomal location In bacteria, coordinately controlled genes are often clustered in an operon that is regulated by a single promoter and transcribed in a single mrna molecule In eukaryotic cells, some co-expressed genes are also clustered near one another of the same chromosomes, but each has its own promoter and control elements More commonly, however, these genes are scattered over different chromosomes, but each has the same combination of control elements Copies of the activators recognize these specific control elements and promote simultaneous transcription of the genes, no matter where they are in a genome This coordinated control often occurs in response to chemical signals (ex: hormones) from outside the cell These signals bind to receptor proteins, forming complexes that serves as transcription activators

36 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

37 RNA Processing RNA processing in the nucleus and export of mature RNA to the cytoplasm provide several opportunities for regulating gene expression in eukaryotic cells 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 each cell type control intron-exon choices Fig by binding to regulatory sequences in the primary transcript DNA Exons Ex) The troponin T Troponin T gene gene encodes 2 different proteins Primary RNA transcript RNA splicing mrna or

38 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, allowing them to be translated repeatedly in these cells The mrna life span is determined in part by sequences in the leader and trailer regions Nucleotide sequences that affect how long an mrna remains intact are often found in the untranslated region (UTR) at the 3 end Animation: mrna Degradation

39 Initiation of Translation Translation presents another opportunity for regulating gene expression Occurs most commonly at the initiation stage The initiation of translation of some mrnas can be blocked by regulatory proteins that bind to sequences or structures within the 5 UTR of the mrna This prevents attachment of ribosomes and hence translation Alternatively, translation of all mrnas in a cell may be regulated simultaneously Usually involves activation or inactivation of one or more protein factors required to initiate translation Ex) Translation initiation factors are simultaneously activated in an egg following fertilization Animation: Blocking Translation

40 Protein Processing and Degradation The final opportunity for controlling gene expression occurs after translation Eukaryotic polypeptides must often be processed to yield functional protein molecules These various types of protein processing include cleavage and chemical modifications Ex) Regulatory proteins are commonly activated or inactivated by the reversible addition of phosphate groups The length of time each protein functions in a cell is also strictly regulated by means of selective degradation To mark a particular protein for destruction, the cell often attaches molecules of a small protein called ubiquitin to that protein Giant protein complexes called proteasomes recognize these ubiquitintagged proteins and degrade them Animation: Protein Degradation Animation: Protein Processing

41 Degradation of a Protein by a Proteasome Step 1: Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol Step 2: The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity Fig Step 3: Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol Ubiquitin Proteasome Proteasome and ubiquitin to be recycled Protein to be degraded Ubiquitinated protein Protein entering a proteasome Protein fragments (peptides)

42 Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression

43 Noncoding RNAs and Regulation of Gene Expression Only a small fraction (1.5% in humans) of DNA codes for proteins, rrna, and trna A significant amount of the genome may be transcribed into noncoding RNAs Noncoding RNAs regulate gene expression at two points: mrna translation Chromatin configuration

44 Effects on mrnas by MicroRNAs and Small Interfering RNAs MicroRNAs (mirnas) are small single-stranded RNA molecules that can bind to complementary sequences of mrna mirnas are formed from longer RNA precursors that fold back on themselves, forming one or more double-stranded hairpin structures, each held together by hydrogen bonds After each hairpin is cut away from the precursor, it is trimmed by an enzyme called a Dicer into a short, double-stranded fragment of ~20 nucleotide pairs One of the two strands is degraded, while the other strand (the mirna) forms a complex with one or more proteins The mirna allows this complex to bind to any mrna molecule with a complementary sequence The mirna-protein complex then either degrades the target mrna or blocks its translation The expression of an estimated 1/3 of all human genes may be regulated by mirnas

45 Step 1: An enzyme cuts each hairpin from the primary mirna transcript Step 2: A second enzyme called Dicer trims the loop and the single-stranded ends from the hairpin (cuts are made at the arrows) Step 3: One strand of the double-stranded RNA is degraded The other strand (mirna) than forms a complex with one or more proteins Step 4: The mirna in the complex can bind to any target mrna that contains at least 6 bases of complementary sequence Step 5: If mirna and mrna bases are complementary all along their length, the mrna is degraded (left) Fig Hairpin 5 3 (a) Primary mirna transcript mirna mirna Hydrogen bond Dicer mirnaprotein complex If the match is less complete, translation is blocked (right) mrna degraded Translation blocked (b) Generation and function of mirnas

46 Small Interfering RNAs Gene expression can also be blocked by RNA molecules called small interfering RNAs (sirnas) The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) sirnas and mirnas are similar but form from different RNA precursors mirna is formed from a single hairpin in a precursor RNA sirnas are formed from much longer double-stranded RNA molecules, each which gives rise to many sirnas

47 Chromatin Remodeling and Silencing of Transcription by Small RNAs Small RNA molecules can also cause remodeling of chromatin structure sirnas play a role in heterochromatin formation and can block large regions of the chromosome An RNA transcript produced from DNA is copied into doublestranded RNA, which is then processed into several sirnas These sirnas associate with a complex of proteins, which then recruit enzymes that modify the chromatin, turning it into the highly condensed heterochromatin Small RNAs may also block transcription of specific genes

48 Concept 18.4: A program of differential gene expression leads to the different cell types in a multicellular organism

49 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 this developmental program, producing cells of different types that form these higher-level structures

50 A Genetic Program for Embryonic Development The transformation from zygote to adult results from 3 interrelated processes: Cell division The zygote gives rise to a large number of cells through a succession of mitotic cell division Cell differentiation These daughter cells then become Fig specialized in structure and function Morphogenesis These different types of cells are organized into tissues and organs in a particular 3-dimensional arrangement that give an organism its shape (a) Fertilized eggs of a frog (b) Newly hatched tadpole

51 Differential gene expression results from genes being regulated differently in each cell type Materials placed into an egg by the mother set up a sequential program of gene regulation that is carried out as cells divide This program makes the cells become different from each other in a coordinated fashion

52 Cytoplasmic Determinants and Inductive Signals Two sources of information tell a cell which genes to express at any given time during embryonic development An egg s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg Fig a These substances include cytoplasmic determinants, 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 Zygote Sperm Two-celled embryo Fertilization Mitotic cell division Unfertilized egg cell Nucle Two different cytoplasmic determinants (a) Cytoplasmic determinants in the egg

53 Induction 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 Gene expression is therefore altered in these cells Thus, interactions between cells induce differentiation of specialized cell types Fig b Early embryo (32 cells) NUCLEUS Signal transduction pathway Animation: Cell Signaling Signal receptor Signal molecule (inducer) (b) Induction by nearby cells

54 Sequential Regulation of Gene Expression During Cellular Differentiation The term determination refers to the events that lead to the observable differentiation of a cell Once a cell has undergone determination, it is irreversibly committed to its final fate If a committed cell is experimentally placed in another location in the embryo, it will still differentiate into the cell type that is its normal fate Determination precedes differentiation Observable cellular differentiation is marked by the expression of genes for tissue-specific proteins These proteins are found only in a specific cell type and give the cell its characteristic structure and function

55 Differentiation of Skeletal Muscle Cells We can look at the differentiation of skeletal muscle cells as an example: Muscle cells develop from embryonic precursor cells that have the potential to develop into a number of cell types, including cartilage and fat cells Once determination occurs, these cells are called myoblasts Myoblasts produce muscle-specific proteins and eventually differentiate to form skeletal muscle cells MyoD is one of several master regulatory genes that produce proteins that commit the cell to becoming skeletal muscle This gene encodes MyoD protein, a transcription factor that binds to enhancers of various target genes and stimulates their expression Then, secondary transcription factors activate the genes for proteins such as myosin and actin that confer the unique properties of skeletal muscle cells

56 Step 1: Determination - Signals from other cells lead to activation of the master regulatory gene myod, allowing the cell to make MyoD protein, which acts as an activator The cell is now called a myoblast and is irreversibly committed to becoming a skeletal muscle cell Step 2: Differentiation - MyoD protein stimulates the myod gene further and activates genes encoding for other muscle-specific transcription factors These transcription factors activate genes for muscle proteins like myosin and actin MyoD also turns on genes that block the cell cycle, thus stopping cell division The nondividing myoblasts fuse to become mature multinucleate muscle cells, also called muscle fibers Fig Nucleus Embryonic precursor cell Myoblast (determined) Master regulatory gene myod DNA Part of a muscle fiber (fully differentiated cell) OFF mrna MyoD protein (transcription factor) Other muscle-specific genes OFF OFF mrna mrna mrna mrna MyoD Another transcription factor Myosin, other muscle proteins, and cell cycle blocking proteins

57 Pattern Formation: Setting Up the Body Plan For differentiated cells and tissues to function effectively in the organism as a whole, the organism s body plan (its 3-D arrangement) must be established and superimposed on the differentiation process 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 The three major axes of a bilaterally symmetrical animal include head and tail, right and left sides, and back and front The molecular cues that control pattern formation are collectively known as positional information These cytoplasmic determinants and inductive signals tell a cell its location relative to the body axes and to neighboring cells

58 Pattern Formation in Drosophila 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

59 The Life Cycle of Drosophila In Drosophila, cytoplasmic determinants in the unfertilized egg provide positional information for the placement of anterior-posterior and dorsalventral axes even before fertilization This egg develops in the female s ovary, surrounded by ovarian cells called nurse cells and follicle cells These support cells supply the egg with nutrients, mrnas, and other substances needed for development and make the egg shell After fertilization, the embryo develops into a segmented larva with three larval stages

60 1) The yellow egg is surrounded by other cells that form a structure called the follicle within one of the mother s ovaries 2)Nurse cells shrink as they supply nutrients and mrnas to the developing egg, which grows larger Eventually, the mature egg fills Fig b the egg shell that is secreted by the follicle cells 3)The egg is fertilized within the mother and then laid Egg cell developing within ovarian follicle Unfertilized egg Fertilized egg Nurse cell Depleted nurse cells Follicle cell Nucleus Egg cell Egg shell Fertilization Laying of egg 4-5) Embryonic development forms Embryonic development a larva that goes through 3 stages The 3 rd stage forms a cocoon (not shown), within which the larva metamorphoses into the adult shown 4 5 Segmented embryo Larval stage Body segments (b) Development from egg to larva Hatching 0.1 mm

61 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 These scientists studied mutant flies with developmental defects that led to extra wings or legs in the wrong places They located these mutations on the fly s genetic map, thus connecting developmental abnormalities to specific genes Fig Their research supplied the first concrete evidence that genes somehow direct developmental processes These genes that control pattern formation in the late embryo, larva, and adult are called homeotic genes Eye Antenna Wild type Leg Mutant

62 Thirty years later, Nüsslein-Volhard and Wieschaus set out to identify all the genes that affect segment formation in Drosophila 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 The researchers were able to group these segmentation genes by general function, to map them, and to clone many of them for further study in the lab

63 Axis Establishment Maternal effect genes encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila When these genes are mutant in the mother, any offspring display the mutant phenotype regardless of the offspring s own genotype These maternal effect genes are also called egg-polarity genes because they control orientation of the egg and consequently, that of the fly One group of these genes sets up the anterior-posterior axis of the embryo, while a second group establishes the dorsal-ventral axis Mutations in these maternal effect genes are generally embryonic lethals Animation: Development of Head-Tail Axis in Fruit Flies

64 The Bicoid Gene 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 This phenotype suggested that the product of the mother s bicoid gene is essential for setting up the anterior end of the fly and therefore might be concentrated at the future anterior end of the embryo This hypothesis is an example of the Fig a morphogen gradient hypothesis, in which gradients of substances called EXPERIMENT Head morphogens establish an embryo s axes and other features of its form Tail T1 T2 T3 A1 A2 A3 A4 A5 Wild-type larva A6 A7 A8 Tail Tail A8 Mutant larva (bicoid) A7 A6 A7 A8

65 Experiment: many embryos and larvae with defects in their body patterns were obtained Some of these defects were due to mutations in the mother s genes, including the bicoid ( two-tailed ) gene, which resulted in larvae with two tails and no head The researchers hypothesized that bicoid normally codes for a morphogen specifying the head (anterior) end of the embryo To test this hypothesis, they used molecular techniques to determine where the mrna and protein encoded by this gene were found in the fertilized egg and early embryo Results: bicoid mrna (dark blue) was confined to the anterior end of the Fig b unfertilized egg Later in development, Bicoid protein was seen to be concentrated in cells at the anterior end of the embryo RESULTS Conclusion: the results support the hypothesis that Bicoid protein is a morphogen specifying formation of headspecific structures 100 µm Bicoid mrna in mature unfertilized egg Fertilization, translation of bicoid mrna Anterior end Bicoid protein in early embryo

66 Importance of Bicoid Research The bicoid research is important for three reasons: It identified a specific protein required for some early steps in pattern formation This helped us understand how different regions of the egg can give rise to cells that go down different developmental pathways It increased understanding of the mother s role in embryo development It demonstrated the key developmental principle that a gradient of molecules can determine polarity and position in the embryo In Drosophila, gradients of specific proteins determine the posterior and anterior ends, as well as the dorsal-ventral axis Positional information later establishes a specific number of correctly oriented segments and triggers the formation of each segment s characteristic structures The pattern of the adult is abnormal when the genes operating in this final step are abnormal

67 Concept 18.5: Cancer results from genetic changes that affect cell cycle control

68 Cancer and Gene Regulation The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development Cancer can be caused by mutations to genes that regulate cell growth and division The agents of these changes can be random spontaneous mutation, or they may be caused by environmental influences, including chemical carcinogens and X-rays Tumor viruses can also cause cancer in animals, including humans Ex) Human papillomaviruses (HPV) are associated with cervical cancer

69 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 An oncogene usually arises from a genetic change that leads to an increase either in the amount of the gene s protein product or in the activity of each protein molecule Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

70 Conversion of Proto-Oncogenes to Oncogenes Genetic changes that convert proto-oncogenes to oncogenes fall into 3 main categories: Movement of DNA (translocation) 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 protooncogene Fig in the cell Point mutation in a control element or in the proto-oncogene itself, causing an increase in gene expression Proto-oncogene DNA Translocation or transposition: Gene amplification: Point mutation: within a control element within the gene New promoter Oncogene Oncogene Normal growthstimulating protein in excess Normal growth-stimulating protein in excess Normal growthstimulating protein in excess Hyperactive or degradationresistant protein

71 Tumor-Suppressor Genes Cells also contain genes known as tumor-suppressor genes whose normal products inhibit cell division The proteins they encode help prevent uncontrolled cell growth Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset The protein products of tumor-suppressor genes have various functions: Repair damaged DNA, preventing the cell from accumulating cancer-causing mutations Control adhesion of cells to one another or to the extracellular matrix, which is crucial in normal tissues and often absent in cancers Inhibit the cell cycle in the cell-signaling pathway

72 Interference with Normal Cell-Signaling Pathways The proteins encoded by many proto-oncogenes and tumorsuppressor genes are components of cell-signaling pathways The products of 2 key genes, ras proto-oncogene and the p53 tumor-suppressor gene, can be examined in order to elucidate what goes wrong with the functioning of these proteins in cancer cells Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division

73 The Ras Protein The Ras (named for rat sarcoma) protein is a G protein that relays a signal from a growth factor receptor on the plasma membrane to a cascade of protein kinases The cellular response at the end of the pathway is the synthesis of a protein that stimulates the cell cycle Normally, this pathway will not operate unless triggered by the appropriate growth factor Certain mutations in the ras gene can lead to production of a hyperactive Ras protein that triggers the kinase cascade even in the absence of growth factor Results in increased cell division

74 The normal cell cycle-stimulating pathway is triggered by a growth factor (1) that binds to its receptor (2) in the plasma membrane The signal is relayed to a G protein (3) called Ras Ras is active when GTP is bound to it Ras passes the signal to a series of protein kinases (4) The last kinase (5) activates a transcription activator that turns on one or more genes for proteins that stimulate the cell cycle Results in excessive cell division that may cause cancer Fig a 2 1 Growth factor Receptor G protein GTP Ras Protein kinases (phosphorylation cascade) 5 Ras GTP Transcription factor (activator) DNA MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own Gene expression NUCLEUS Protein that stimulates the cell cycle (a) Cell cycle stimulating pathway

75 The p53 Gene If the DNA of a cell is damaged, another signaling pathway blocks the cell cycle until the damage has been repaired Thus, the genes for components of this pathway act as tumor-suppressor genes One is these genes, called the p53 gene, encodes a specific transcription factor that promotes the synthesis of cell cycle-inhibiting proteins Activates a gene called p21 whose product halts the cell cycle by binding to cyclin-dependent kinases, thus allowing time for DNA repair Also turns on genes directly involved in DNA repair When DNA damage is irreparable, p53 activates suicide genes whose proteins cause cell death by apoptosis A mutation that knocks out the p53 gene can lead to excessive cell growth and cancer

76 In the normal cell cycle-inhibiting pathway, DNA damage (1) is an intracellular signal that is passed via protein kinases (2) and leads to activation of p53 (3) Activated p53 promotes transcription of the gene for a protein that inhibits the cell cycle Fig b This suppression ensures that damaged DNA is not replicated UV light 2 Protein kinases 3 Active form of p53 MUTATION Defective or missing transcription factor, such as p53, cannot activate transcription 1 DNA damage in genome DNA Protein that inhibits the cell cycle (b) Cell cycle inhibiting pathway

77 Fig Mutations causing deficiencies in any pathway component can contribute to the development of cancer Increased cell division that may lead to cancer can result if the cell cycle is over-stimulated via the cell cycle-stimulating pathway (a) 2 1 Growth factor Receptor (a) Cell cycle stimulating pathway UV light DNA damage in genome G protein GTP Ras Protein kinases (phosphorylation cascade) 2 Protein kinases 3 DNA 5 Active form of p53 GTP Ras Transcription factor (activator) DNA MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own Gene expression Protein that stimulates the cell cycle NUCLEUS MUTATION Defective or missing transcription factor, such as p53, cannot activate transcription A similar effect can be seen if the mutation affects the cell cycle-inhibiting pathway (b) (b) Cell cycle inhibiting pathway Protein overexpressed Protein that inhibits the cell cycle EFFECTS OF MUTATIONS Protein absent Cell cycle overstimulated Increased cell division Cell cycle not inhibited

78 The Multistep Model of Cancer Development Multiple mutations are generally needed for full-fledged cancer The longer we live, the more mutations we accumulate This may help explain why the incidence of cancer increases greatly 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

79 The model of a multistep path to cancer is well-supported by studies of colorectal cancer Like most cancers, colorectal cancer develops gradually The 1 st sign is often a polyp, made up of cells that look normal but divide unusually frequently The tumor grows and may eventually become malignant, spreading to other tissues The development of this malignant tumor is caused by a gradual accumulation Fig of mutations that convert proto-oncogenes to oncogenes and knock out tumorsuppressor genes A ras oncogene and a mutated p53 tumor-suppressor gene are often involved Colon EFFECTS OF MUTATIONS Colon wall 1 Loss of tumorsuppressor gene APC (or other) 2 Activation of ras oncogene 4 Loss of tumor-suppressor gene p53 Normal colon epithelial cells Small benign growth (polyp) 3 Loss of tumor-suppressor gene DCC Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma)

80 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

81 You should now be able to: 1. Explain the concept of an operon and the function of the operator, repressor, and corepressor 2. Explain the adaptive advantage of grouping bacterial genes into an operon 3. Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control

82 4. Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription 5. Define control elements and explain how they influence transcription 6. Explain the role of promoters, enhancers, activators, and repressors in transcription control

83 7. Explain how eukaryotic genes can be coordinately expressed 8. Describe the roles played by small RNAs on gene expression 9. Explain why determination precedes differentiation 10. Describe two sources of information that instruct a cell to express genes at the appropriate time

84 11. Explain how maternal effect genes affect polarity and development in Drosophila embryos 12. Explain how mutations in tumor-suppressor genes can contribute to cancer 13. Describe the effects of mutations to the p53 and ras genes