Regulation of Gene Expression

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Chapter 18

• Regulation of Gene Expression

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Overview Conducting the Genetic Orchestra

• Prokaryotes 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

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Eye
Leg
Antenna
Wild type
Mutant
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Bacteria regulate their gene expression

• Feedback mechanisms allow control over metabolism
  so that cells produce only the products needed at
  that time (with some limitations)
• This metabolic control occurs on two levels
• 1. adjusting activity of metabolic enzymes
• 2. regulating genes that encode metabolic
  enzymes

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Regulation of a Metabolic Pathway
Precursor
Feedback inhibition
trpE gene
Enzyme 1

• In the pathway for tryptophan synthesis,
• an abundance of tryptophan can both
• inhibit the activity of the first enzyme
  in the pathway (feedback inhibition),
  a rapid response, and
• repress expression of the genes for all the
  enzymes needed for the pathway, a
  longer-term response.

Regulation of gene expression
trpD gene
trpC gene
Enzyme 2
trpB gene
Enzyme 3
trpA gene
Tryptophan
(b) Regulation of enzyme production

• Regulation of
• enzyme activity

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• In bacteria, genes are often clustered into
  operons, composed of
• 1. an operator, an on-off switch
• 2. a promoter
• 3. genes for metabolic enzymes
• An operon can be switched off by a protein called
  a repressor
• binds only to the operator

trp operon
Promoter
Promoter
Genes of operon (5)
DNA
trpB
trpA
trpR
trpE
trpD
trpC
Operator
Regulatory gene
no tryptophan
Stop codon
RNA polymerase
Start codon
3
repressor inactive
mRNA 5
mRNA
5
E
D
C
B
A
operon ON
Polypeptides (5) that make up enzymes for
tryptophan synthesis
Protein
Repressor
(inactive)
Tryptophan absent, repressor inactive, operon on
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Operons

• 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
• repressible enzymes usually function in anabolic
  pathways
• cells suspend production of an end product that
  is not needed

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The trp operon
operon OFF
repressor active
tryptophan
DNA
DNA
No RNA made
mRNA
mRNA
Protein
Active repressor
Protein
Active repressor
corepressor- a small molecule that cooperates
with a repressor to switch an operon off
Tryptophan (corepressor)
Tryptophan (corepressor)
Tryptophan present, repressor active, operon off
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Operons

• 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
• repressible enzymes usually function in anabolic
  pathways
• cells suspend production of an end product that
  is not needed
• An inducible operon-
• is one that is usually off
  
  a molecule, an inducer,
  inactivates the repressor turns on
  transcription
• the classic example of an inducible operon is the
  lac operon
• inducible enzymes usually function in catabolic
  pathways
• cells only produce enzymes when theres a
  nutrient that needs to be broken down

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The lac operon
Promoter
Regulatory gene
Operator
lacl
lacZ
DNA
No RNA made
no lactose
3
mRNA
RNA polymerase
repressor active
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operon OFF
Active repressor
Protein
Lactose absent, repressor active, operon off
The lac repressor is innately active, and in the
absence of lactose it switches off the operon by
binding to the operator.
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Allolactose, an isomer of lactose, derepresses
the operon by inactivating the repressor. In this
way, the enzymes for lactose utilization are
induced.
lac operon
3 genes
DNA
lacl
lacZ
lacY
lacA
RNA polymerase
3
mRNA
mRNA 5
5
Permease
Transacetylase
?-Galactosidase
Protein
E.Coli uses 3 enzymes to take up and metabolize
lactose
Inactive repressor
Allolactose (inducer)
ß-galactosidase hydrolyzes lactose to glucose
and galactose permease transports lactose into
the cell transacetylase function in lactose
metabolism is still unclear
Lactose present, repressor inactive, operon on
inducer- a small molecule that
inactivates the repressor
operon ON
repressor inactive
lactose
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Gene Regulation

• Regulation of the trp and lac operons involves
  negative control of genes because operons are
  switched off by the active form of the repressor
• Some operons are also subject to positive control
  through a stimulatory activator protein,
  such as catabolite activator protein
  (CAP)
• The lac operon is under dual control
• negative control by the lac repressor
• positive control by CAP

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Positive Control

• When glucose (a preferred food source of E. coli
  ) is scarce,
  the lac operon is
  activated by the binding of CAP

Promoter
DNA
lacl
lacZ
RNA polymerase can bind and transcribe
Operator
CAP-binding site
glucose
cAMP
Active CAP
cAMP
Inactive lac repressor
Inactive CAP
Lactose present, glucose scarce (cAMP level
high) abundant lac mRNA synthesized
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When glucose levels increase, CAP detaches from
the lac operon, turning it off
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA polymerase cant bind
Inactive CAP
Inactive lac repressor
Lactose present, glucose present (cAMP level
low) little lac mRNA synthesized
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REVIEW
Repressible Operon
Genes not expressed
Genes expressed
Promoter
Genes
Operator
Active repressor corepressor bound
Inactive repressor no corepressor present
Corepressor
Inducible Operon
Genes not expressed
Genes expressed
Promoter
Operator
Genes
Active repressor no inducer present
Inactive repressor inducer bound
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Eukaryotic Genomes

• Two features of eukaryotic genomes are
  a major
  information-processing challenge
• 1. the typical eukaryotic genome is much larger
  than that of a prokaryotic cell
• 2. cell specialization limits the expression of
  many genes to specific cells

The DNA-protein complex, called chromatin,
is
ordered into higher structural levels than the
DNA-protein complex in prokaryotes
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Differential Gene Expression

• Almost all the cells in an organism are
  genetically identical
• Differences between cell types result from
• differential gene expression,
• the expression of different genes by cells with
  the same genome
• Errors in gene expression can lead to diseases
  including cancer

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Gene Expression
Signal
NUCLEUS
Chromatin
Chromatin modificationDNA unpacking
involvinghistone acetylation andDNA
demethylation
1

• Gene expression is regulated at many stages

DNA
Gene availablefor transcription
Gene
2
most important control point
Transcription
Exon
RNA
Primary transcript
Intron
3
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
4
5
Translation
Degradationof mRNA
Polypeptide
Protein processing, suchas cleavage and
chemical modification
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6
Active protein
Degradationof protein
Transport to cellulardestination
Cellular function (suchas enzymatic
activity,structural support)
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Histones
1

• Genes within highly packed heterochromatin
  (highly condensed areas)
  are usually not expressed
• Chemical modifications to histones and DNA of
  chromatin influence both
  chromatin structure and gene
  expression
• histone acetylation-
• acetyl groups (-COCH3) are attached to
  positively charged lysines in histone tails
• This process seems to loosen chromatin structure,
  thereby promoting the initiation of transcription

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2
3
5
4
6
Histone tails
DNA double helix
Amino acids available for chemical modification
Histone tails protrude outward from a nucleosome
Unacetylated histones
Acetylated histones
Acetylation of histone tails promotes loose
chromatin structure that permits transcription
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DNA Methylation
1

• DNA methylation-
• the addition of methyl groups to certain bases
  in DNA, is associated with reduced
  transcription in some species
• DNA methylation can cause long-term inactivation
  of genes in cellular differentiation
• In genomic imprinting, methylation turns off
  either the maternal or paternal alleles of
  certain genes at the start of development

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Summary- Chromatin Modifications
1

• Although the chromatin modifications just
  discussed do not alter DNA sequence, they may be
  passed to future generations of cells
• The inheritance of traits transmitted by
  mechanisms not directly involving the
  nucleotide sequence is called
• epigenetic inheritance
• 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

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Eukaryotic Gene
2 3

• Associated with most eukaryotic genes are
  multiple control elements, segments
  of noncoding DNA that help regulate transcription
  by binding certain proteins
• Control elements the proteins they bind are
  critical to the precise
  regulation of gene expression in different cell
  types

Enhancer (distal control elements)
Proximal control elements
Poly-A signal sequence
Termination region
Exon
Intron
Exon
Exon
Intron
DNA
Upstream
Downstream
Promoter
Transcription
Poly-A signal
Exon
Intron
Exon
Intron
Exon
Primary RNA transcript (pre-mRNA)
Cleaved 3 end of primary transcript
1
5
2
RNA processing Cap and tail added introns
excised and exons spliced together
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Intron RNA
5
4
Coding segment
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mRNA
3
Start codon
Stop codon
5 UTR (untranslated region)
5 Cap
Poly-A tail
3 UTR (untranslated region)
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2

• To initiate transcription, eukaryotic RNA
  polymerase requires the
• assistance of proteins called transcription
  factors (TFs)
• General TFs are essential for the transcription
  of all protein-coding genes
• In eukaryotes, high levels of transcription of
  particular genes depend on control
  elements interacting with specific TFs

• proximal control elements-
• are located close to the promoter
• distal control elements-
  groups of which are called
  enhancers, may be
  far away from a gene
  or even in an intron
• activator-
• specific TF,
  a protein that binds
  to an enhancer
  stimulates transcription of a gene

Activator proteins bind to distal control
elements grouped as an enhancer in the DNA. This
enhancer has three binding sites.
A DNA-bending protein brings the bound activators
closer to the promoter. Other transcription
factors, mediator proteins, and RNA polymerase
are nearby.
The activators bind to certain general
transcription factors and mediator proteins,
helping them form an active transcription
initiation complex on the promoter.
repressor-
specific TF,

inhibit expression of a gene
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Combinatorial Control of Gene Activation
2
Enhancer
Promoter
Controlelements
Albumin gene

• A particular combination of
• control elements can activate
• transcription only when the
• appropriate activator proteins
• are present

Crystallingene
LIVER CELLNUCLEUS
LENS CELLNUCLEUS
Availableactivators
Availableactivators
Albumin genenot expressed
Albumin geneexpressed
Crystallin genenot expressed
Crystallin geneexpressed
(a) Liver cell
(b) Lens cell
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Coordinately Controlled Genes
2

• Unlike the genes of a prokaryotic operon,
  each of the co-expressed eukaryotic genes
  has a promoter and control elements
• These genes can be scattered over
  different chromosomes, but each has the
  same combination of control elements
• Copies of the activators recognize
  specific control elements and promote
  simultaneous transcription of the genes

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Mechanisms of Post-Transcriptional Regulation
3-6

• Transcription alone does not account for gene
  expression
• More and more examples are being found of
  regulatory mechanisms that
  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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Alternative RNA Splicing
3
Exons
DNA
Troponin T gene
Primary RNA transcript
different mRNA molecules are produced
from the same primary transcript,
depending on which RNA segments
are treated as exons/introns
RNA splicing
or
mRNA
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mRNA Degradation
4

• 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
• Nucleotide sequences that influence the lifespan
  of mRNA in eukaryotes reside in the untranslated
  region (UTR) at
  the 3 end of the molecule

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Initiation of Translation
5

• The initiation of translation of selected mRNAs
• can be blocked by regulatory proteins that bind
  to specific sequences or structures of the mRNA
• Alternatively, translation of all mRNAs in a
  cell may be regulated simultaneously
• For example, translation initiation factors are
  simultaneously activated in an egg
  following fertilization

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Protein Processing and Degradation
6

• After translation, various types of protein
  processing,
  including cleavage and the addition
  of chemical groups, are subject to control

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proteasomes- are giant protein complexes that
bind protein molecules and degrade them
2
3
4
5
Enzymatic components of the proteasome cut the
protein into small peptides, which can be further
degraded by other enzymes in the cytosol.
Multiple ubiquitin molecules are attached to a
protein by enzymes in the cytosol.
The ubiquitin-tagged protein is recognized by a
proteasome, which unfolds the protein
and sequesters it within a central cavity.
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Proteasome and ubiquitin to be recycled
Ubiquitin
Proteasome
Protein to be degraded
Ubiquitinated protein
Protein fragments (peptides)
Protein entering a proteasome
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REVIEW
Transcription
Chromatin modification
Genes in highly compactedchromatin are
generally nottranscribed.
Regulation of transcription initiationDNA
control elements in enhancers bindspecific
transcription factors.
Histone acetylation seemsto loosen chromatin
structure,enhancing transcription.
Bending of the DNA enables activators tocontact
proteins at the promoter, initiatingtranscription
.
DNA methylation generallyreduces transcription.
Coordinate regulation
Enhancer forliver-specific genes
Enhancer forlens-specific genes
Chromatin modification
Transcription
RNA processing
Alternative RNA splicing
RNA processing
Primary RNAtranscript
Translation
mRNAdegradation
mRNA
or
Protein processingand degradation
Translation
Initiation of translation can be controlledvia
regulation of initiation factors.
mRNA degradation
Each mRNA has a characteristic life
span,determined in part bysequences in the 5?
and3? UTRs.
Protein processing and degradation
Protein processing anddegradation by
proteasomesare subject to regulation.
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Noncoding RNAs play multiple roles in
controlling gene expression

• Only a small fraction of DNA codes for proteins,
  and a very small
  fraction of the non-protein-coding DNA consists
  of genes for RNA such as rRNA and tRNA
• A significant amount of the genome may be
  transcribed into noncoding RNAs (ncRNAs)
• Noncoding RNAs regulate gene expression at two
  points
• mRNA translation
• chromatin configuration

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Hairpin
Hydrogenbond
miRNA
Dicer
5?
3?
(a) Primary miRNA transcript
miRNA
miRNA-proteincomplex

• MicroRNAs (miRNAs)-
• small single-stranded RNA molecules
  that can bind to mRNA
• These can degrade mRNA or block its translation

mRNA degraded
Translation blocked
(b) Generation and function of miRNAs
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• 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
• In some yeasts siRNAs play a role in
  heterochromatin formation
  and can block large regions of the chromosome
• RNA-based mechanisms may also block transcription
  of single genes

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REVIEW
Chromatin modification
Small or large noncoding RNAs canpromote the
formation of heterochromatinin certain regions,
blocking transcription.
Chromatin modification
Transcription
Translation
RNA processing
miRNA or siRNA can block the translationof
specific mRNAs.
mRNAdegradation
Translation
Protein processingand degradation
mRNA degradation
miRNA or siRNA can target specificmRNAs for
destruction.
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