chapter 18 regulation of gene expression. the basics prokaryotes and eukaryotes alter gene...
TRANSCRIPT
Chapter 18Regulation of Gene
Expression
The BasicsProkaryotes and 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
RNA molecules play many roles in regulating gene expression in eukaryotes
18.1 Bacterial Gene Regulation
Bacteria respond to changes in environment
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
Fig. 18-2
Regulationof geneexpression
trpE gene
trpD gene
trpC gene
trpB gene
trpA gene
(b) Regulation of enzyme production
(a) Regulation of enzyme activity
Enzyme 1
Enzyme 2
Enzyme 3
Tryptophan
Precursor
Feedbackinhibition
OperonsA 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
RepressorsThe 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
Repressors• 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
E. coli and Tryptophan• By default the trp operon is on and the genes for
tryptophan synthesis are transcribed
• BUT 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
Fig. 18-3
Polypeptide subunits that make upenzymes for tryptophan synthesis
(b) Tryptophan present, repressor active, operon off
Tryptophan(corepressor)
(a) Tryptophan absent, repressor inactive, operon on
No RNA made
Activerepressor
mRNA
Protein
DNA
DNA
mRNA 5
Protein Inactiverepressor
RNApolymerase
Regulatorygene
Promoter Promoter
trp operon
Genes of operon
OperatorStop codonStart codon
mRNA
trpA
5
3
trpR trpE trpD trpC trpB
ABCDE
Fig. 18-3a
Polypeptide subunits that make upenzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
DNA
mRNA 5
Protein Inactiverepressor
RNApolymerase
Regulatorygene
Promoter Promoter
trp operon
Genes of operon
OperatorStop codonStart codon
mRNA
trpA
5
3
trpR trpE trpD trpC trpB
ABCDE
Fig. 18-3b-1
(b) Tryptophan present, repressor active, operon off
Tryptophan(corepressor)
No RNA made
Activerepressor
mRNA
Protein
DNA
Fig. 18-3b-2
(b) Tryptophan present, repressor active, operon off
Tryptophan(corepressor)
No RNA made
Activerepressor
mRNA
Protein
DNA
trp OperonRepressible operon
one that is usually onbinding of a repressor to the operator shuts off
transcription
E. Coli lac OperonAllows bacteria to use lactose (sugar) as food
Genes are turned off by repressors
Genes are turned on by presence of lactose in the cell
lac operonInducible operon
one that is usually offa molecule called an inducer inactivates the
repressor and turns on transcription
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
Fig. 18-4
(b) Lactose present, repressor inactive, operon on
(a) Lactose absent, repressor active, operon off
mRNA
Protein
DNA
DNA
mRNA 5
ProteinActiverepressor
RNApolymerase
Regulatorygene
Promoter
Operator
mRNA5
3
Inactiverepressor
Allolactose(inducer)
5
3
NoRNAmade
RNApolymerase
Permease Transacetylase
lac operon
-Galactosidase
lacYlacZ lacAlacI
lacI lacZ
Fig. 18-4a
(a) Lactose absent, repressor active, operon off
DNA
ProteinActiverepressor
RNApolymerase
Regulatorygene
Promoter
Operator
mRNA5
3
NoRNAmade
lacI lacZ
Fig. 18-4b
(b) Lactose present, repressor inactive, operon on
mRNA
Protein
DNA
mRNA 5
Inactiverepressor
Allolactose(inducer)
5
3
RNApolymerase
Permease Transacetylase
lac operon
-Galactosidase
lacYlacZ lacAlacI
Inducible enzymes Inducible enzymes usually function in catabolic
pathways (breakdown complex substances, release energy)
their synthesis is induced by a chemical signal
Repressible enzymes Repressible enzymes usually function in
anabolic pathways (synthesis of large molecules, require energy)
their synthesis is repressed by high levels of the end product
Positive Gene RegulationSome operons are also subject to positive
control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription
Positive Gene RegulationWhen glucose (a preferred food source of E. coli)
is scarce, CAP is activated by binding with cyclic AMP
Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription
Positive Gene RegulationWhen glucose levels increase, CAP detaches
from the lac operon, and transcription returns to a normal rate
CAP helps regulate other operons that encode enzymes used in catabolic pathways
18.2 Eukaryotic Gene Regulation
Eukaryotic Gene Regulation
Differential Gene Expression
Regulation of Chromatin Structure
Histone modification
DNA Methylation and Genomic Imprinting
Epigenetic Inheritance
Eukaryotic Gene Regulation
All organisms must regulate which genes are expressed at any given time
In multicellular organisms gene expression is essential for cell specialization
Differential Gene Expression
Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome
Regulation of Chromatin Structure
Genes within highly packed heterochromatin are usually not expressed
Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression
Histone ModificationsHistone acetylation: acetyl groups are attached
to positively charged lysines in histone tailsLoosens chromatin structure, promoting the
initiation of transcription
Addition of methyl groups (methylation) can condense chromatin
Addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin
Fig. 18-7
Histonetails
DNAdouble helix
(a) Histone tails protrude outward from a nucleosome
Acetylated histones
Aminoacidsavailablefor chemicalmodification
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
DNA MethlylationDNA methylation can cause long-term
inactivation of genes in cellular differentiation
Causes chromatin to condense
In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development
Epigenetic InheritanceAlthough 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
DNA and Histone Methylation Activity
Regulation of Transcription Initiation
Chromatin-modifying enzymes initial control of gene expression by making a
region of DNA either more or less able to bind the transcription machinery
Control Elementssegments of noncoding DNA that help regulate
transcription by binding certain proteins
Fig. 18-8-1
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
ExonExon ExonIntron Intron
Fig. 18-8-2
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
Exon Exon ExonIntronIntron Cleaved 3 endof primarytranscript
Primary RNAtranscript
Poly-Asignal
Transcription
5
ExonExon ExonIntron Intron
Fig. 18-8-3
Enhancer(distal control elements)
Proximalcontrol elements
Poly-A signalsequence
Terminationregion
DownstreamPromoter
UpstreamDNA
ExonExon ExonIntron Intron
Exon Exon ExonIntronIntron Cleaved 3 endof primarytranscript
Primary RNAtranscript
Poly-Asignal
Transcription
5
RNA processing
Intron RNA
Coding segment
mRNA
5 Cap 5 UTRStart
codonStop
codon 3 UTR Poly-Atail
3
Review: Transcription Factors
To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors
General transcription factors: essential for all protein-coding genes
Specific transcription factors: depend on interaction of control elements
Transcription Regulators: Control Elements
Proximal control elements located close to the promoter
Distal control elementsmay be far away from a gene or even located in an
intronSome groups of distal control elements are called
enhancersEngancers are gene-specific – only control one gene
Even more control of gene expression
Activators
Repressors
ActivatorsProtein that binds to an enhancer and stimulates
transcription of a gene
Bound activators cause mediator proteins to interact with proteins at the promoter
Fig. 18-9-1
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Fig. 18-9-2
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Group ofmediator proteins
DNA-bendingprotein
Generaltranscriptionfactors
Fig. 18-9-3
Enhancer TATAbox
PromoterActivators
DNAGene
Distal controlelement
Group ofmediator proteins
DNA-bendingprotein
Generaltranscriptionfactors
RNApolymerase II
RNApolymerase II
Transcriptioninitiation complex RNA synthesis
RepressorsInhibiting expression of a particular gene
Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription
The Role of Activators and Repressors
Control transcription initiationBlock activator sitesControl chromatin structureHistone acetylationHistone deacetylation
Silencing
Combinatorial Control of Gene Activation
• Combination of control elements (activators and repressors) can activate transcription only when the appropriate activator proteins are present• Cell specialization (lens of the eye vs. liver)
Fig. 18-10
Controlelements
Enhancer
Availableactivators
Albumin gene
(b) Lens cell
Crystallin geneexpressed
Availableactivators
LENS CELLNUCLEUS
LIVER CELLNUCLEUS
Crystallin gene
Promoter
(a) Liver cell
Crystallin genenot expressed
Albumin geneexpressed
Albumin genenot expressed
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
Post-Transcriptional Regulation
RNA processing
mRNA degradation
Initiation of Translation
Protein Processing and Degradation
Post-Transcriptional Regulation: RNA
ProcessingAlternative 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
Fig. 18-11
or
RNA splicing
mRNA
PrimaryRNAtranscript
Troponin T gene
Exons
DNA
Post-Transcriptional Regulation: 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 mRNABacteria can quickly alter expressed genes
because mRNA breaks down quickly
The mRNA life span is determined in part by sequences in the leader and trailer regions
Post-Transcriptional Regulation: Initiation of
TranslationSelected mRNAs can be blocked by regulatory
proteins that bind to sequences on the mRNA
Translation of ALL mRNA molecules can also be regulatedtranslation initiation factors are simultaneously
activated in an egg following fertilization
Post-Transcriptional Regulation: Protein Processing
and DegradingProtein processing, including cleavage and the
addition of chemical groups, are subject to control
Length of time each protein is active is controlledProteasomes: giant protein complexes that bind
protein molecules and degrade themUbiquitin: tags protein for degradation
Fig. 18-12
Proteasomeand ubiquitinto be recycledProteasome
Proteinfragments(peptides)Protein entering a
proteasome
Ubiquitinatedprotein
Protein tobe degraded
Ubiquitin
18.3 Noncoding RNAs play multiple roles in
controlling gene expression
Noncoding RNAsOnly a small fraction of DNA codes for proteins,
rRNA, and tRNAA significant amount of the genome may be
transcribed into noncoding RNAs
Noncoding RNAs regulate gene expression at two pointsmRNA translationchromatin configuration
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
MicroRNAs (miRNAs)Forms from double-stranded RNA (the other
strand is the degraded)
Forms a complex with protein
Bonds with another RNA molecule with a complementary sequenceDegrade mRNABlock transcription
Fig. 18-13
miRNA-proteincomplex(a) Primary miRNA transcript
Translation blocked
Hydrogenbond
(b) Generation and function of miRNAs
Hairpin miRNA
miRNA
Dicer
3
mRNA degraded
5
Inhibition of Gene Expression by RNA
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
Chromatin remodeling and silencing of transcription
by Small RNAssiRNAs play a role in heterochromatin formation
and can block large regions of the chromosome
Small RNAs may also block transcription of specific genes
Now…On to the REALLY interesting part of gene
regulation:
Now…On to the REALLY interesting part of gene regulation:
CELL DIFFERENTIATION IN MULTICELLULAR ORGANISMS !!!!!!!
18.4 A program of differential gene
expression leads to the different cell types in a multicellular organism
Fertilization…and so it begins…
Fertilization…and so it begins…
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
DevelopmentThe transformation from zygote to adult results
from cell divisioncell differentiation: cells become specialized in
structure and functionMorphogenesis: gives organisms shape; drives
the arrangement of specialized cells
Before cells differentiate, they are:pluripotent (stem cells in adults)Totipotent (stem cells in embryos)
How long do you think this takes?
(a) Fertilized eggs of a frog (b) Newly hatched tadpole
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 divideCytoplasmic determinants
Cytoplasmic DeterminantsAn egg’s cytoplasm contains RNA, proteins, and
other substances that are distributed unevenly in the unfertilized egg
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
Fig. 18-15
(b) Induction by nearby cells(a) Cytoplasmic determinants in the egg
Two differentcytoplasmicdeterminants
Unfertilized egg cell
Sperm
Fertilization
Zygote
Mitoticcell division
Two-celledembryo
Signalmolecule(inducer)
Signaltransductionpathway
Early embryo(32 cells)
Nucleus
NUCLEUS
Signalreceptor
Developmental Signaling: Induction
The other important source of developmental information is the environment around the cell, especially signals from nearby embryonic cells
Induction: signal molecules from embryonic cells cause transcriptional changes in nearby target cells
Thus, interactions between cells induce differentiation of specialized cell types
Step One in Cell Differentiation: Determination
Determination commits a cell to its final fate Irreversible
Determination precedes differentiation
Cell differentiation is marked by the production of tissue-specific proteins
More about determinationTissue specific proteins
Step 1: mRNA for the tissue specific proteins are produced
Different genes will be expressed in different tissues
Tissue Differentiation: Muscle
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 other various target genes
Fig. 18-16-3
Embryonicprecursor cell
Nucleus
OFF
DNA
Master regulatory gene myoD Other muscle-specific genes
OFF
OFFmRNA
MyoD protein(transcriptionfactor)
Myoblast(determined)
mRNA mRNA mRNA mRNA
Myosin, othermuscle proteins,and cell cycle–blocking proteinsPart of a muscle fiber
(fully differentiated cell)
MyoD Anothertranscriptionfactor
Now…
Now……we need to figure out where all these newly differentiated tissues will go in the developing organism
Now……we need to figure out where all these newly differentiated tissues will go in the developing organism
Aaaaand…what better organism to study than the…
Drosophila melanogaster
!!!!
duh…
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
Why study Drosophila?Combining anatomical, genetic, and biochemical
approaches, researchers have discovered developmental principles common to many other species, including humans
The Drosophila Life CycleCytoplasmic determinants in the unfertilized egg
determine the axes before fertilization
After fertilization, the embryo develops into a segmented larva
Fig. 18-17bFollicle cell
Nucleus
Eggcell
Nurse cell
Egg celldeveloping withinovarian follicle
Unfertilized egg
Fertilized egg
Depletednurse cells
Eggshell
FertilizationLaying of egg
Bodysegments
Embryonicdevelopment
Hatching
0.1 mm
Segmentedembryo
Larval stage
(b) Development from egg to larva
1
2
3
4
5
Fig. 18-17a
ThoraxHead Abdomen
0.5 mm
Dorsal
Ventral
Right
Posterior
LeftAnteriorBODY
AXES
(a) Adult
Genetic Analysis of Early Development: Scientific
InquiryEdward B. Lewis, Christiane Nüsslein-Volhard, and
Eric Wieschaus won a Nobel 1995 Prize for decoding pattern formation in Drosophila
Genetic Analysis of Early Development: Scientific
InquiryEdward B. Lewis
Studied flies with developmental defects that led to extra wings, eyes in the wrong places
demonstrated that genes direct the developmental process – homeotic genes – control pattern formation
Fig. 18-18
Antenna
MutantWild type
Eye
Leg
Genetic Analysis of Early Development: Scientific
InquiryNüsslein-Volhard and Wieschaus studied segment
formationCreated mutants, conducted breeding experiments,
and looked for corresponding genes
Breeding experiments were complicated by embryonic lethals, embryos with lethal mutations
They found 1200 genes essential for pattern formation and 120 of the genes for segmentation alone
Axis Establishment: Maternal Effect Genes
Maternal effect genes encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila If mother has mutation, offspring will have
mutation regardless of genotype.
Axis Establishment: Maternal Effect Genes
Maternal effect genes are also called egg-polarity genes because they control orientation of the egg and consequently the flyAnterior/posterior axis, dorsal/ventral axis
Maternal effect gene mutations are usually lethal
Bicoid: A Morphogen Determining Head
Structures
Work with a partner to outline (in your notes) the experiment on the bicoid gene and morphogens
Include: purpose, hypothesis, procedure and conclusion
Follow Up Questions1. What does the bicoid gene determine?
2. What is a morphogen?
3. What is the function of nurse cells? Where do they come from?
4. Where does the mRNA for the bicoid gene originate in the unfertilized egg?
5. Predict what would happen if you injected bicoid mRNA into the anterior end of an egg from a female mutant for bicoid.
Fig. 18-19a
T1 T2T3
A1 A2 A3 A4 A5 A6A7
A8
A8A7 A6 A7
Tail
TailTail
Head
Wild-type larva
Mutant larva (bicoid)
EXPERIMENT
A8
Fig. 18-19b
Fertilization,translationof bicoidmRNA Bicoid protein in early
embryo
Anterior endBicoid mRNA in matureunfertilized egg
100 µm
RESULTS
Fig. 18-19c
bicoid mRNA
Nurse cells
Egg
Developing egg Bicoid mRNA in matureunfertilized egg
Bicoid proteinin early embryo
CONCLUSION
Who cares?
Who cares?The bicoid research is important for three
reasons: Identified a specific protein required for some
early steps in pattern formation Increased understanding of the mother’s role in
embryo developmentDemonstrated a key developmental principle that
a gradient of molecules can determine polarity and position in the embryo
Concept 18.5: Cancer results from
genetic changes that affect cell cycle
control
CancerThe gene regulation systems that go wrong
during cancer are the very same systems involved in embryonic development
Genes Associated With Cancer
Cancer can be caused by mutations to genes that regulate cell growth and division
Tumor viruses can cause cancer in animals including humans
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
Proto-oncogene OncogeneAbnormal stimulation of the cell cycle
Proto-oncogenes Oncogenes
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 elementscauses an increase in gene expression
Tumor-suppressor genesHelp prevent uncontrolled cell growth
Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset
Tumor-suppressor proteinsRepair damaged DNA
Control cell adhesion
Inhibit the cell cycle in the cell-signaling pathway
Interference with Normal Cell-Signaling Pathways
ras proto-oncogene
p53 tumor-suppressor gene
Fig. 18-21a
Receptor
Growthfactor
G protein GTP
Ras
GTP
Ras
Protein kinases(phosphorylationcascade)
Transcriptionfactor (activator)
DNA
HyperactiveRas protein(product ofoncogene)issuessignalson its own
MUTATION
NUCLEUS
Gene expression
Protein thatstimulatesthe cell cycle
(a) Cell cycle–stimulating pathway
11
3
4
5
2
Fig. 18-21b
MUTATIONProtein kinases
DNA
DNA damagein genome
Defective ormissingtranscriptionfactor, suchas p53, cannotactivatetranscription
Protein thatinhibitsthe cell cycle
Activeformof p53
UVlight
(b) Cell cycle–inhibiting pathway
2
3
1
Fig. 18-21c
(c) Effects of mutations
EFFECTS OF MUTATIONS
Cell cycle notinhibited
Protein absent
Increased celldivision
Proteinoverexpressed
Cell cycleoverstimulated
Suppression of the cell cycle by p53
p53 prevents a cell from passing on mutations due to DNA damage
Mutations in the p53 gene prevent suppression of the cell cycle
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
Inherited Predisposition and Other Factors Contributing to
Cancer
Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes
Colorectal cancer inherited mutations in the tumor-suppressor gene
adenomatous polyposis coli
Inherited breast cancers: at least half are linked to mutations in the BRCA1 or BRCA2
Fig. 18-22a
Colon
Colon wall
Normal colonepithelial cells
Fig. 18-22b
Loss of tumor-suppressor geneAPC (or other)
Small benigngrowth (polyp)
1
Fig. 18-22c
Activation ofras oncogene
Loss oftumor-suppressorgene DCC
Larger benigngrowth (adenoma)
2
3
Fig. 18-22d
Malignant tumor(carcinoma)
Loss oftumor-suppressorgene p53
Additionalmutations5
4