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How Genes are Controlled
Muse 2009
CONTROL OF GENE EXPRESSION
– Gene expression is the overall process of information flow from genes to proteins
– Mainly controlled at the level of transcription
– A gene that is “turned on” is being transcribed to produce mRNA that is translated to make its corresponding protein
– Organisms respond to environmental changes by controlling gene expression
11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response to
environmental changes
11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response
to environmental changes– An operon is a group of genes under
coordinated control in bacteria
– The lactose (lac) operon includes
– Three adjacent genes for lactose-utilization enzymes
– Promoter sequence where RNA polymerase binds
– Operator sequence is where a repressor can bind and block RNA polymerase action
11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response
to environmental changes
Regulation of the lac operon– Regulatory gene codes for a repressor protein
– In the absence of lactose, the repressor binds to the operator and prevents RNA polymerase action
– Lactose inactivates the repressor, so the operator is unblocked
11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response
to environmental changes– Types of operon control
– Inducible operon (lac operon)– Active repressor binds to the operator
– Inducer (lactose) binds to and inactivates the repressor
– Repressible operon (trp operon)– Repressor is initially inactive
– Corepressor (tryptophan) binds to the repressor and makes it active
– For many operons, activators enhance RNA polymerase binding to the promoter (ie NAC)
DNA
DNA
RNA polymerasecannot attach to promoter
Lactose-utilization genesPromoter OperatorRegulatorygene
OPERON
Protein
mRNA
Inactiverepressor
Lactose Enzymes for lactose utilization
RNA polymerasebound to promoter
Operon turned on (lactose inactivates repressor)
mRNA
Activerepressor
Operon turned off (lactose absent)
Protein
DNA
RNA polymerasecannot attach to promoter
Lactose-utilization genesPromoter OperatorRegulatorygene
OPERON
mRNA
Activerepressor
Operon turned off (lactose absent)
Protein
DNA
Protein
Inactiverepressor
Lactose Enzymes for lactose utilization
RNA polymerasebound to promoter
Operon turned on (lactose inactivates repressor)
mRNA
DNA
Inactiverepressor
Activerepressor
Inactiverepressor
Activerepressor
Lactose
Promoter
Tryptophan
Operator Gene
lac operon trp operon
11.2 Differentiation results from the expression of different combination
genes
– Differentiation involves cell specialization, in both structure and function
– Differentiation is controlled by turning specific sets of genes on or off
11.3 DNA packing in eukaryotic chromosomes helps regulate gene
expression– Eukaryotic chromosomes undergo multiple levels of folding and
coiling, called DNA packing – Nucleosomes are formed when DNA is wrapped around
histone proteins– “Beads on a string” appearance– Each bead includes DNA plus 8 histone molecules– String is the linker DNA that connects nucleosomes
– Tight helical fiber is a coiling of the nucleosome string– Supercoil is a coiling of the tight helical fiber– Metaphase chromosome represents the highest level of
packing– DNA packing can prevent transcription
11.3 DNA packing in eukaryotic chromosomes helps regulate
gene expression
Animation: DNA Packing
DNA double helix(2-nm diameter)
“Beads ona string”
Linker
Histones
Metaphasechromosome
Tight helical fiber(30-nm diameter)
Nucleosome(10-nm diameter)
Supercoil(300-nm diameter)
700 nm
11.4 In female mammals, one X chromosome is inactive in each somatic cell
– X-chromosome inactivation
– In female mammals, one of the two X chromosomes is highly compacted and transcriptionally inactive
– Random inactivation of either the maternal or paternal chromosome
– Occurs early in embryonic development and all cellular descendants have the same inactivated chromosome
– Inactivated X chromosome is called a Barr body
– Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats
Two cell populationsin adult
X chromosomes
Early embryo
Allele forblack fur
Inactive X
Black furAllele fororange fur
Orange fur
Cell divisionand random
X chromosomeinactivation Active X
Inactive X
Active X
11.5 Complex assemblies of proteins control
eukaryotic transcription
– Eukaryotic genes – Each gene has its own promoter and terminator
– Are usually switched off and require activators to be turned on
– Are controlled by interactions between numerous regulatory proteins and control sequences
11.5 Complex assemblies of proteins control
eukaryotic transcription
– Regulatory proteins that bind to control sequences
– Transcription factors promote RNA polymerase binding to the promoter
– Activator proteins bind to DNA enhancers and interact with other transcription factors
– Silencers are repressors that inhibit transcription
– Control sequences
– Promoter
– Enhancer
– Related genes located on different chromosomes can be controlled by similar enhancer sequences
11.5 Complex assemblies of proteins control eukaryotic
transcription
Animation: Initiation of Transcription
Regions on the DNA called enhancers control genes that may be quitedistant.Enhancer binding proteins may have multiple controls built into them
Enhancers
Otherproteins
DNA
Transcriptionfactors
Activatorproteins
RNA polymerase
Promoter
Gene
Bendingof DNA
Transcription
11.6 Eukaryotic RNA may be spliced in more than one way
– Alternative RNA splicing – Production of different mRNAs from the same
transcript
– Results in production of more than one polypeptide from the same gene
– Can involve removal of an exon with the introns on either side
Animation: RNA Processing
1
or
Exons
DNA
RNA splicing
RNAtranscript
mRNA
2 3 4 5
1 2 3 4 5
1 2 4 51 2 3 5
11.7 Small RNAs play multiple roles in
controlling gene expression
– RNA interference (RNAi)
– Prevents expression of a gene by interfering with translation of its RNA product
– Involves binding of small, complementary RNAs to mRNA molecules
– Leads to degradation of mRNA or inhibition of translation
– MicroRNA
– Single-stranded chain about 20 nucleotides long
– Binds to protein complex
– MicroRNA + protein complex binds to complementary mRNA to interfere with protein production
11.7 Small RNAs play multiple roles in controlling gene expression
Animation: Blocking Translation
Animation: mRNA Degradation
Anti-sense RNA and snRNAi
miRNA 1
Translation blockedORmRNA degraded
Target mRNA
Protein
miRNA-proteincomplex
2
3 4
11.8 Translation and later stages of gene expression are also subject
to regulation
– Control of gene expression also occurs with – Breakdown of mRNA
– Initiation of translation
– Protein activation
– Protein breakdown
Folding ofpolypeptide andformation ofS—S linkages
Initial polypeptide(inactive)
Folded polypeptide(inactive)
Active formof insulin
Cleavage
Disulfide bonds
Quartenary structure
11.9 Review: Multiple mechanisms regulate
gene expression in eukaryotes
– Many possible control points exist; a given gene may be subject to only a few of these
– Chromosome changes (1)– DNA unpacking
– Control of transcription (2)– Regulatory proteins and control sequences
– Control of RNA processing– Addition of 5’ cap and 3’ poly-A tail (3)
– Splicing (4)
– Flow through nuclear envelope (5)
11.9 Review: Multiple mechanisms regulate
gene expression in eukaryotes
– Many possible control points exist; a given gene may be subject to only a few of these
– Breakdown of mRNA (6)
– Control of translation (7)
– Control after translation – Cleavage/modification/activation of proteins (8)
– Breakdown of protein (9)
– Applying Your KnowledgeFor each of the following, determine whether an increase or decrease in the amount of gene product is expected
– The mRNA fails to receive a poly-A tail during processing in the nucleus
– The mRNA becomes more stable and lasts twice as long in the cell cytoplasm
– The region of the chromatin containing the gene becomes tightly compacted
– An enzyme required to cleave and activate the protein product is missing
11.9 Review: Multiple mechanisms regulate
gene expression in eukaryotes
– Applying Your KnowledgeFor each of the following, determine whether an increase or decrease in the amount of gene product is expected
– The mRNA fails to receive a poly-A tail during processing in the nucleus ---------
– The mRNA becomes more stable and lasts twice as long in the cell cytoplasm ++++++
– The region of the chromatin containing the gene becomes tightly compacted --------
– An enzyme required to cleave and activate the protein product is missing +++++ feedback
11.9 Review: Multiple mechanisms regulate
gene expression in eukaryotes
11.9 Review: Multiple mechanisms regulate gene expression in
eukaryotes
Animation: Protein Processing
Animation: Protein Degradation
NUCLEUS
DNA unpackingOther changes to DNA
Addition of cap and tail
Chromosome
Gene
RNA transcript
GeneTranscription
Intron
Exon
Splicing
CapmRNA in nucleus
Tail
Flow throughnuclear envelope
Broken-downmRNA
CYTOPLASM
Breakdown of mRNA
Translation
mRNA in cytoplasm
Broken-downprotein
Cleavage / modification /activation
Breakdown of protein
Polypeptide
Active protein
NUCLEUS
DNA unpackingOther changes to DNA
Addition of cap and tail
Chromosome
Gene
RNA transcript
GeneTranscription
Intron
Exon
Splicing
CapmRNA in nucleus
Tail
Flow throughnuclear envelope
Broken-downmRNA
CYTOPLASM
Breakdown of mRNA
Translation
mRNA in cytoplasm
Broken-downprotein
Cleavage / modification /activation
Breakdown of protein
Polypeptide
Active protein
11.10 Cascades of gene expression direct the development
of an animal
– Role of gene expression in fruit fly development
– Orientation from head to tail
– Maternal mRNAs present in the egg are translated and influence formation of head to tail axis
– Segmentation of the body
– Protein products from one set of genes activate other sets of genes to divide the body into segments
– Production of adult features
– Homeotic genes are master control genes that determine the anatomy of the body, specifying structures that will develop in each segment
Animation: Development of Head-Tail Axis in Fruit Flies
11.10 Cascades of gene expression direct the development of an
animal
Animation: Cell Signaling
Head of a normal fruit fly
Antenna
Eye
Head of a developmental mutant
Leg
Egg cellwithin ovarianfollicle
Follicle cells
“Head”mRNA
Proteinsignal
Egg cell
Gene expression1
Cascades ofgene expression2
Embryo Bodysegments
Adult fly
Gene expression
3
4
DNA microarrays test for the transcription of many genes at
once– DNA microarray
– Contains DNA sequences arranged on a grid
– Used to test for transcription– mRNA from a specific cell type is isolated
– Fluorescent cDNA is produced from the mRNA
– cDNA is applied to the microarray
– Unbound cDNA is washed off
– Complementary cDNA is detected by fluorescence
cDNA
Nonfluorescent spotFluorescent
spot
Actual size(6,400 genes)
Each well contains DNAfrom a particular gene
DNA microarray
mRNAisolated
DNA of anexpressed gene
DNA of anunexpressed gene
Reverse transcriptaseand fluorescent DNAnucleotides
1
cDNA madefrom mRNA
2
cDNA appliedto wells
3
UnboundcDNA rinsedaway
4
Global transcription analysis
11.12 Signal transduction pathways convert messages received at the cell surface to responses within
the cell – Signal transduction pathway is a series of molecular changes that converts a signal at the cell’s surface to a response within the cell
– Signal molecule is released by a signaling cell
– Signal molecule binds to a receptor on the surface of a target cell
11.12 Signal transduction pathways convert messages received at the cell surface to responses within
the cell – Relay proteins are activated in a series of reactions
– A transcription factor is activated and enters the nucleus
– Specific genes are transcribed to initiate a cellular response
Animation: Signal Transduction Pathways
Animation: Overview of Cell Signaling
Signaling cell
DNA
Nucleus
Transcriptionfactor(activated)
Signaling molecule Plasma
membraneReceptorprotein
Relayproteins
TranscriptionmRNA
Newprotein
Translation
Target cell
2
1
3
4
5
6
CLONING OF PLANTS AND ANIMALS
Hello Dolly!
11.14 Plant cloning shows that differentiated cells may retain all of their genetic
potential
– Most differentiated cells retain a full set of genes, even though only a subset may be expressed
– Evidence is available from – Plant cloning
– A root cell can divide to form an adult plant
– Animal limb regeneration
– Remaining cells divide to form replacement structures
– Involved dedifferentiation followed by redifferentiation into specialized cells
Root ofcarrot plant
Root cells culturedin nutrient medium
Cell divisionin culture
Single cell
Plantlet Adult plant
11.15 Nuclear transplantation can be used to clone animals
– Nuclear transplantation– Replacing the nucleus of an egg cell or zygote
with a nucleus from an adult somatic cell
– Early embryo (blastocyst) can be used in – Reproductive cloning
– Implant embryo in surrogate mother for development
– New animal is genetically identical to nuclear donor
– Therapeutic cloning
– Remove embryonic stem cells and grow in culture for medical treatments
– Induce stem cells to differentiate
Removenucleusfrom eggcell
Implant blastocyst insurrogate mother
Add somatic cellfrom adult donor
Donorcell
Remove embryonicstem cells fromblastocyst andgrow in culture
Reproductivecloning
Nucleus fromdonor cell
Grow in cultureto produce anearly embryo(blastocyst)
Therapeuticcloning
Clone ofdonor is born
Induce stemcells to formspecialized cells
Removenucleusfrom eggcell
Add somatic cellfrom adult donor
Donorcell Nucleus from
donor cell
Grow in cultureto produce anearly embryo(blastocyst)
Implant blastocyst insurrogate mother
Remove embryonicstem cells fromblastocyst andgrow in culture
Reproductivecloning
Therapeuticcloning
Clone ofdonor is born
Induce stemcells to formspecialized cells
Reproductive cloning has valuable applications, but human reproductive
cloning raises ethical issues– Cloned animals can show differences from their
parent due to a variety of influences during development
– Reproductive cloning is used to produce animals with desirable traits
– Agricultural products
– Therapeutic agents
– Restoring endangered animals
– Human reproductive cloning raises ethical concerns
CAT1 CC
11.17 CONNECTION: Therapeutic cloning can produce stem cells with great
medical potential– Stem cells can be induced to give rise to
differentiated cells – Embryonic stem cells can differentiate into a
variety of types
– Adult stem cells can give rise to many but not all types of cells
– Therapeutic cloning can supply cells to treat human diseases
– Research continues into ways to use and produce stem cells
Blood cells
Different types ofdifferentiated cells
Nerve cells
Adult stemcells in bone
marrow
Different cultureconditions
Culturedembryonicstem cells
Heart muscle cells
THE GENETIC BASIS OF CANCER
11.18 Cancer results from mutations in genes that control cell division
– Mutations in two types of genes can cause cancer
– Oncogenes– Proto-oncogenes normally promote cell division
– Mutations to oncogenes enhance activity
– Tumor-suppressor genes– Normally inhibit cell division
– Mutations inactivate the genes and allow uncontrolled division to occur
11.18 Cancer results from mutations in genes that control cell division
– Oncogenes
– Promote cancer when present in a single copy
– Can be viral genes inserted into host chromosomes (src, ras)
– Can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation
– Converting a proto-oncogene to an oncogene can occur by– Mutation causing increased protein activity
– Increased number of gene copies causing more protein to be produced
– Change in location putting the gene under control of new promoter for increased transcription
11.18 Cancer results from mutations in genes that control cell division
– Tumor-suppressor genes– Promote cancer when both copies are mutated
Mutation withinthe gene
Hyperactivegrowth-stimulatingprotein in normalamount
Proto-oncogene DNA
Multiple copiesof the gene
Gene moved tonew DNA locus,
under new controls
Oncogene New promoter
Normal growth-stimulatingprotein in excess
Normal growth-stimulatingprotein in excess
Mutated tumor-suppressor geneTumor-suppressor gene
Defective,nonfunctioningprotein
Normalgrowth-inhibitingprotein
Cell divisionunder control
Cell division notunder control
11.19 Multiple genetic changes underlie the
development of cancer
– Four or more somatic mutations are usually required to produce a cancer cell
– One possible scenario for colorectal cancer includes
– Activation of an oncogene increases cell division
– Inactivation of tumor suppressor gene causes formation of a benign tumor
– Additional mutations lead to a malignant tumor
1
Colon wall
Cellularchanges:
DNAchanges:
Oncogeneactivated
Increasedcell division
Tumor-suppressorgene inactivated
Growth of polyp
Second tumor-suppressor geneinactivated
Growth of malignanttumor (carcinoma)
2 3
Chromosomes 1mutation
Normalcell
4mutations
3mutations
2mutations
Malignantcell
11.20 Faulty proteins can interfere with normal signal transduction
pathways
– Path producing a product that stimulates cell division
• Product of ras proto-oncogene relays a signal when growth hormone binds to receptor
• Product of ras oncogene relays the signal in the absence of hormone binding, leading to uncontrolled growth
11.20 Faulty proteins can interfere with
normal signal transduction pathways
– Path producing a product that inhibits cell division– Product of p53 tumor-suppressor gene is a
transcription factor
– p53 transcription factor normally activates genes for factors that stop cell division
– In the absence of functional p53, cell division continues because the inhibitory protein is not produced
Growth factor
Protein thatStimulatescell division
Translation
Nucleus
DNA
Target cell
Normal productof ras gene
Receptor
Relayproteins
Transcriptionfactor(activated)
Hyperactiverelay protein(product ofras oncogene)issues signalson its own
Transcription
Growth-inhibiting factor
Protein thatinhibitscell division
Translation
Normal productof p53 gene
Receptor
Relayproteins
Transcriptionfactor(activated)
Nonfunctional transcriptionfactor (product of faulty p53tumor-suppressor gene) cannot trigger transcription
Transcription
Protein absent(cell divisionnot inhibited)
Lifestyle choices can reduce the risk of cancer
– Carcinogens are cancer-causing agents that damage DNA and promote cell division
– X-rays and ultraviolet radiation
– Tobacco
– Healthy lifestyle choices– Avoiding carcinogens
– Avoiding fat and including foods with fiber and antioxidants
– Regular medical checkups
Wrap up and Review
• Bacteria arrange multiple genes into operons and control promoters with repressors and activators.
• Eukaryotes have multiple ways to control genes.
Regulatorygene
Promoter
DNA
Gene 1Operator
A typical operon
Gene 2Gene 3
Encodes repressorthat in active formattaches to operator
RNApolymerasebinding site
Switches operonon or off
Nucleus fromdonor cell
Early embryoresulting fromnuclear trans-plantation
Surrogatemother
Cloneof donor
Whole Organism Cloning
Nucleus fromdonor cell
Early embryoresulting fromnuclear trans-plantation
Embryonicstem cellsin culture
Specializedcells
Therapeutic cloning
Generegulation
(a)prokaryoticgenes oftengrouped into
in eukaryotesmay involve when
abnormalmay lead to
can produce
is a normal gene thatcan be mutated to an
oncogene
can cause
(c)
(g)(f)
multiple mRNAsper genetranscription
femalemammals
(e)
(d)
(b)
operons
areswitchedon/off by
in activeform binds to
controlled byprotein called
occurs inare proteinsthat promote
§ Explain how prokaryotic gene control occurs in the operon
§ Describe the control points in expression of a eukaryotic gene
§ Describe DNA packing and explain how it is related to gene expression
§ Explain how alternative RNA splicing and microRNAs affect gene expression
§ Compare and contrast the control mechanisms for prokaryotic and eukaryotic genes
You should now be able to
§ Distinguish between terms in the following groups: promoter—operator; oncogene—tumor suppressor gene; reproductive cloning— therapeutic cloning
§ Define the following terms: Barr body, carcinogen, DNA microarray, homeotic gene; stem cell; X-chromosome inactivation
§ Describe the process of signal transduction, explain how it relates to yeast mating, and explain how it is disrupted in cancer development
You should now be able to
§ Explain how cascades of gene expression affect development
§ Compare and contrast techniques of plant and animal cloning
§ Describe the types of mutations that can lead to cancer
§ Identify lifestyle choices that can reduce cancer risk
You should now be able to