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Regulation of Gene Expression-Chapter 15
• Prokaryotes and eukaryotes alter gene expression in response to their changing environment
• In multicellular eukaryotes, gene expression also regulates development and is responsible for differences in cell types
• Protein/DNA, RNA/DNA, and RNA/RNA interactions all play roles in regulating gene expression in eukaryotes
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Bacteria often respond to environmental change by regulating transcription
• Natural selection has favored bacteria that produce only the products needed by that cell
• A cell can regulate the production of enzymes by feedback inhibition or by gene regulation
• Which of these forms of regulation saves more energy? Which can respond to changes more quickly?
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LE 18-20
Regulation of enzymeactivity
Regulation of enzymeproduction
Enzyme 1
Regulation of gene expression
Enzyme 2
Enzyme 3
Enzyme 4
Enzyme 5
Gene 2
Gene 1
Gene 3
Gene 4
Gene 5
Tryptophan
Precursor
Feedbackinhibition
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The Operon Model
• Gene expression in bacteria is controlled by the operon model
• Jacob and Monod (1961)
• Operon elements-repressor gene; operator; structural gene(s)
• Inducible and repressible operons
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Operons: The Basic Concept
• A group of functionally related genes can be coordinately controlled 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
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Repressible and Inducible Operons: Two Types of Negative Gene Regulation
• A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription
• The lac operon is an inducible operon
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• Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal
• Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product
• Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor
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The repressible operon (trp operon)
• Shuts down a biochemical pathway when the end product of the pathway build up.
• Normal status-transcription occurs because the operator is unblocked.
• End product of the pathway (co-repressor) alters shape of the repressor protein so that it binds to the operator blocking transcription of the structural genes
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LE 18-21a
Promoter Promoter
DNA trpR
Regulatorygene
RNApolymerase
mRNA
3
5
Protein Inactiverepressor
Tryptophan absent, repressor inactive, operon on
mRNA 5
trpE trpD trpC trpB trpA
OperatorStart codonStop codon
trp operon
Genes of operon
E
Polypeptides that make upenzymes for tryptophan synthesis
D C B A
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LE 18-21b_1
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
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LE 18-21b_2
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
No RNA made
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• By default the trp operon is on and the genes for tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp repressor protein, which then 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
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Inducible operons
• Activates breakdown of a substrate when the substrate becomes available.
• Normal status-transcription does not occurs because the operator is blocked by the repressor protein.
• Substrate (inducer) alters shape of the repressor protein so that does not bind to the operator allowing transcription of the structural genes to occur
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• The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose
• By itself, the lac repressor is active and switches the lac operon off
• A molecule called an inducer inactivates the repressor to turn the lac operon on
• For the lac operon, the inducer is allolactose, formed from lactose that enters the cell
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LE 18-22a
DNA lacl
Regulatorygene
mRNA
5
3
RNApolymerase
ProteinActiverepressor
NoRNAmade
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off
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LE 18-22b
DNA lacl
mRNA5
3
lac operon
Lactose present, repressor inactive, operon on
lacZ lacY lacA
RNApolymerase
mRNA 5
Protein
Allolactose(inducer)
Inactiverepressor
-Galactosidase Permease Transacetylase
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Positive Gene Regulation
• The lac operon can be turned on or off by the presence/absence of the inducer molecule (lactose)
• CAP and CAMP can also regulate the rate of transcription of the lac operon (volume control versus on/off control)
• If both glucose and lactose are present, the bacteria will preferentially use glucose
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LE 18-23a
DNA
cAMP
lacl
CAP-binding site
Promoter
ActiveCAP
InactiveCAP
RNApolymerasecan bindand transcribe
Operator
lacZ
Inactive lacrepressor
Lactose present, glucose scarce (cAMP level high): abundant lacmRNA synthesized
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LE 18-23b
DNA lacl
CAP-binding site
Promoter
RNApolymerasecan’t bind
Operator
lacZ
Inactive lacrepressor
InactiveCAP
Lactose present, glucose present (cAMP level low): little lacmRNA synthesized
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Positive Gene Regulation
• E. coli will preferentially use glucose when it is present in the environment
• When glucose is scarce, CAP (catabolite activator protein) acts as an activator of transcription
• CAP is activated by binding with cyclic AMP (cAMP)
• Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription
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• When glucose levels increase, CAP detaches from the lac operon, and transcription proceeds at a very low rate, even if lactose is present
• CAP helps regulate other operons that encode enzymes used in catabolic pathways
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Differential Gene Expression in Eukaryotes
• Almost all the cells in an multicellular organism are genetically identical
• Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome
• In a given cell, usually about 20% of its genes are active at a given time (rest turned off)
• Errors in gene expression can lead to diseases including cancer
• Gene expression is regulated at many stages in eukaryotes (in addition to regulation at the transcriptional level which bacteria also do)
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Regulation of chromatin Structure:
• DNA is packed into an elaborate complex known as chromatin
• The basic unit of chromtain is the nucleosome• Histone proteins help maintain nucleosome
structure• The structural organization of chromatin packs
DNA into a compact form and also helps regulate gene expression in several ways
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Fig. 16-21b
30-nm fiber
Chromatid (700 nm)
Loops Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber Looped domains (300-nm fiber)
Metaphase chromosome
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Fig. 16-21a
DNA double helix (2 nm in diameter)
Nucleosome(10 nm in diameter)
Histones Histone tailH1
DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)
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Chromatin Packing in Eukaryotes
• Loosely packed DNA (euchromatin) is usually transcribed
• Tightly packed DNA (heterochromatin) is not usually transcribed
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Regulation of Chromatin Structure
• Chemical modifications to histones and DNA of chromatin influence both chromatin structure (tightly packed versus loosely packed) and gene expression
• Chemical modification of histones include acetylation and methylation
• Chemical modification of DNA include methylation
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Histone Modifications
• In histone acetylation, acetyl groups (-COCH3)are attached to positively charged lysines in histone tails
• This process loosens chromatin structure, thereby promoting the initiation of transcription
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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
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DNA Methylation
• DNA methylation, the addition of methyl groups (CH3) to certain bases in DNA, usually cytosine, is associated with reduced transcription in some species
• DNA methylation can cause long-term inactivation of genes in cellular differentiation
• Barr Bodies• Once methylated, genes usually remain so through
successive cell divisions• After replication, enzymes methylate the correct daughter
strand so that the methylation pattern is inherited
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Epigenetic Inheritance
• Though 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 modifications can be reversed, unlike mutations in DNA sequence
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Epigenetic Inheritance
• Might explain why one twin gets a genetic disease (schizophrenia) and the other does not.
• Alterations in normal DNA methylation patterns are seen in some cancers-these changes can cause inappropriate gene expression in cancer cells.
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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
• Regulation of transcription by enhancers (and their associated control elements) is another important mechanism by which initiation of transcription can be regulated.
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Organization of a Typical Eukaryotic Gene
• Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins
• Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types
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Figure 15.8
DNAUpstream
Enhancer(distal control
elements)
Proximalcontrol
elementsTranscription
start site
Exon Intron Exon
Promoter
Intron Exon
Poly-A signalsequence
Transcriptiontermination
region
Down-stream
Transcription
Exon Intron IntronExon Exon
Poly-Asignal
Primary RNAtranscript(pre-mRNA)
5 Cleaved 3end ofprimarytranscript
Intron RNA
mRNA
RNA processing
Coding segment
3
5 5 3Cap UTRStart
codonStop
codon UTR Poly-Atail
G P P P AAAAAA
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The Roles of Transcription Factors
• To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors. React with proximal control elements of the gene (DNA)
• General transcription factors are essential for the transcription of all protein-coding genes
• If a gene is going to be transcribed at anything but a slow rate, specific transcription factor must also be involved.
• In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors
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• An activator is a protein (a specific transcription factor) that binds to a (distal) control element of an enhancer and stimulates transcription of a gene
• Activators have two domains, one that binds DNA and a second that activates transcription
• Bound activators cause mediator proteins to interact with proteins at the promoter
Enhancers and Specific Transcription Factors
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Figure 15.9
Activationdomain
DNA
DNA-bindingdomain
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• Bound activators are brought into contact with a group of mediator proteins through DNA bending
• The mediator proteins in turn interact with proteins at the promoter
• These protein-protein interactions help to assemble and position the initiation complex on the promoter
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Figure 15.10-3
DNA
EnhancerDistal controlelement
Activators PromoterGene
TATA box
DNA-bendingprotein
Group of mediator proteins
General transcriptionfactors
RNApolymerase II
RNApolymerase II
RNA synthesisTranscriptioninitiation complex
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Why does a liver cell produce albumin and a lens cell crystalline protein?
• All activators needed for high-level expression of the albumin gene are present in liver cells but not lens cells.
• All activators needed for high-level expression of the crystalline gene are present in lens cells but not liver cells.
• The presence of activators in cells may occur at precise times during development or in a particular cell type
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Figure 15.11Albumin gene
Crystallin gene
Promoter
Promoter
(b) LENS CELL NUCLEUS
Availableactivators
Albumin genenot expressed
Crystallin geneexpressed
Crystallin genenot expressed
Albumin geneexpressed
Availableactivators
(a) LIVER CELL NUCLEUS
Controlelements
Enhancer
Enhancer
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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
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Figure 15.UN02
Chromatin modification
Transcription
RNA processing
TranslationmRNAdegradation
Proteinprocessing
and degradation
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RNA Processing
• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns
• In Drosophila-one gene has enough exons that alternative splicing can produce 19,000 different membrane proteins. Each nerve cell has a different membrane protein. Estimates are that 90% of human genes are alternatively spliced.
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Figure 15.12
DNA
PrimaryRNAtranscript
mRNA or
Exons
Troponin T gene
RNA splicing
1 2 3 4 5
1 2 3 5 1 2 4 5
1 2 3 4 5
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Figure 15.UN03
Chromatin modification
Transcription
RNA processing
TranslationmRNAdegradation
Proteinprocessing
and degradation
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mRNA Degradation
• The life span of mRNA molecules in the cytoplasm is important in determining the pattern of protein synthesis in a cell
• Eukaryotic mRNA generally survives longer than prokaryotic mRNA
• Nucleotide sequences that influence the life span of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule
• Noncoding rna’s can cause m-rna to be degraded or have it inhibited (more about that later)
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Protein Processing and Degradation
• After translation, various types of protein processing, including cleavage and chemical modification, are subject to control (pro-insulin insulin)
• The length of time each protein functions in a cell is regulated by means of selective degradation.To mark a particular protein for destruction, the cell commonly attaches molecules of ubiquitin to the protein, which triggers its destruction
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Figure 15.UN06Chromatin modification Transcription
RNA processing
Translation
mRNA degradation Protein processing and degradation
• Protein processing and degradation aresubject to regulation.
• Each mRNA has acharacteristic life span.
• Initiation of translation can be controlledvia regulation of initiation factors.
mRNA or
Primary RNAtranscript
• Alternative RNA splicing:
• The genes in a coordinately controlledgroup all share a combination of controlelements.
• Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors.
Bending ofthe DNAenablesactivators tocontact proteins atthe promoter, initiating transcription.
• Genes in highly compactedchromatin are generally nottranscribed.• Histone acetylation seemsto loosen chromatinstructure,enhancingtranscription.
• DNA methylation generallyreduces transcription.
Chromatin modification
Transcription
RNA processing
mRNAdegradation
Translation
Protein processingand degradation
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Noncoding RNAs play multiple roles in controlling gene expression
• Protein coding DNA only accounts for about 1.5% of the human genome
• Only a small fraction of DNA codes for rRNA, and tRNA. What is the rest of DNA for?
• A significant amount of the genome may be transcribed into noncoding (nc)RNAs (rock star versus backups)
• Noncoding RNAs regulate gene expression at the level of translation by degrading M-rna or blocking its translation
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Effects on mRNAs by MicroRNAs and Small Interfering RNAs
• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to complementary mRNA sequences
• These can degrade the mRNA or block its translation
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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
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• Another class of small RNAs are called small interfering RNAs (siRNAs)
• siRNAs and miRNAs are similar but form from different RNA precursors
• The phenomenon of inhibition of gene expression by siRNAs is called RNA interference (RNAi)
• RNAi may represent the evolution of a genetic control system from a defense mechanism against double stranded RNA virus infection.
• Researchers uses RNAi to knock out genes and study their function.
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Researchers can monitor expression of specific genes
• Cells of a given multicellular organism differ from each other because they express different genes from an identical genome
• The most straightforward way to discover which genes are expressed by cells of interest is to identify the mRNAs being made
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Studying the Expression of Single Genes
• We can detect mRNA in a cell using nucleic acid hybridization, the base pairing of a strand of nucleic acid to its complementary sequence
• The complementary molecule in this case is a short single-stranded DNA or RNA called a nucleic acid probe
• Each probe is labeled with a fluorescent tag to allow visualization
• The technique allows us to see the mRNA in place (in situ) in the intact organism and is thus called in situ hybridization
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Figure 15.14
50 m
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• Genome-wide expression studies can be carried out using DNA microarray assays
• A microarray—also called a DNA chip—contains tiny amounts of many single-stranded DNA fragments affixed to the slide in a grid
• The experiment can identify subsets of genes that are being expressed differently in one sample (breast cancer tissue for example)compared to another (normal tissue).
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Figure 15.17
Genes in redwells expressedin first tissue.
Genes in greenwells expressedin second tissue.
Genes in yellowwells expressedin both tissues.
Genes in blackwells notexpressed in either tissue.
DNA microarray
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Notes for microarray diagram
• Each little dot represent a well in a plate.• Each well has a gene from the two tissues
tested. This plate has 5,760 wells (about 25% of human genes).
• The plate is incubated with probes from the two tissues (one tissues probes are labeled green and the other red).
• Colors for each well indicate whether the gene is expressed in one tissue (red or green), both tissues (yellow) or neither (black)