Chapter 18
Regulation of Gene Expression
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
Wild type Mutant
Eye
AntennaLeg
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
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
PrecursorFeedbackinhibition
In the pathway for tryptophan synthesis, an abundance of tryptophan can both (a)inhibit the activity of the first enzyme in the pathway (feedback inhibition), a rapid response, and(b)repress expression of the genes for all the enzymes needed for the pathway, a longer-term response.
Regulation of a Metabolic Pathway
• 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
Promoter Promoter
DNA trpR
Regulatorygene RNA
polymerase
mRNA
3
5
Protein Repressor
Tryptophan absent, repressor inactive, operon on
mRNA 5
trpE trpD trpC trpB trpA
OperatorStart codonStop codon
trp operon
Genes of operon (5)
E
Polypeptides (5) that make upenzymes for tryptophan synthesis
D C B A
operonON
no tryptophan
repressorinactive
(inactive)
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
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
DNA
Protein
Tryptophan(corepressor)
mRNA
Activerepressor
No RNA made
corepressor- a small molecule that cooperates with a repressor to switch an operon off
The trp operonoperon
OFFtryptophan repressor
active
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 there’s a nutrient that needs to be broken down
DNA lacl
Regulatorygene
mRNA
5
3
RNApolymerase
ProteinActiverepressor
NoRNAmade
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off
The lac operon
The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator.
operonOFF
no lactose
repressoractive
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
Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.
3 genes
E.Coli uses 3 enzymes to take up and metabolize lactose
β-galactosidase: hydrolyzes lactose to glucose and galactosepermease: transports lactose into the celltransacetylase: function in lactose metabolism is still unclear
operonON
lactose repressorinactive
inducer- a small molecule that inactivates the repressor
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
DNA
cAMP
lacl
CAP-binding site
Promoter
ActiveCAP
InactiveCAP
RNApolymerasecan bindand transcribe
Operator
lacZ
Inactive lacrepressor
Lactose present, glucose scarce (cAMP level high):abundant lac mRNA synthesized
• When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP
Positive Control
glucose
cAMP
DNA lacl
CAP-binding site
Promoter
RNApolymerasecan’t bind
Operator
lacZ
Inactive lacrepressor
InactiveCAP
Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
When glucose levels increase, CAP detaches from the lac operon, turning it off
Promoter
Genes
Genes not expressed
Active repressor:no inducer present
Inactive repressor:inducer bound
Genes expressed
Operator
Promoter
Genes
Genes not expressed
Inactive repressor:no corepressor present Corepressor
Active repressor:corepressor bound
Genes expressed
Operator
REVIEWRepressible Operon
Inducible Operon
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
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
Signal
NUCLEUSChromatin
Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation
DNA
Gene
Gene availablefor transcription
RNA ExonPrimary transcript
Transcription
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
TranslationDegradationof mRNA
Polypeptide
Protein processing, suchas cleavage and chemical modification
Active proteinDegradationof protein
Transport to cellulardestination
Cellular function (suchas enzymatic activity,structural support)
Gene Expression
• Gene expression is regulated at many stages
#1
#2
#3
#4
#6
***** most important control point
#5
#6
• 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
Histonetails
Amino acidsavailablefor chemicalmodification
DNAdouble helix
Histone tails protrude outward from a nucleosome
Acetylation of histone tails promotes loose chromatinstructure that permits transcription
Unacetylated histones Acetylated histones
Histones#1
#1
#2
#3
#4
#6
#5
DNA Methylation
• 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
#1
Summary- Chromatin Modifications
• 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
#1
Eukaryotic Gene• 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
Upstream
DNA
Promoter
Exon Intron Exon Intron Exon
DownstreamTranscription
Poly-A signalsequence
Terminationregion
Intron Exon Intron Exon
RNA processing:Cap and tail added;introns excised andexons spliced together
Poly-A signal
Cleaved 3 endof primarytranscript
3
Poly-Atail
3 UTR(untranslated
region)
5 UTR(untranslated
region)
Startcodon
Stopcodon
Coding segment
Intron RNA
5 Cap
mRNA
Primary RNAPrimary RNAtranscripttranscript(pre-mRNA)(pre-mRNA)
5Exon
#2 & #3
#1
#2
#3
#4 #5
#6
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
repressor- specific TF, inhibit expression of a gene
#2
Activator proteins bindto distal control elementsgrouped as an enhancer in the DNA. This enhancer hasthree 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.
Controlelements
Enhancer Promoter
Albumin gene
Crystallingene
LIVER CELLNUCLEUS
Availableactivators
Albumin geneexpressed
Crystallin genenot expressed
(a) Liver cell
LENS CELLNUCLEUS
Availableactivators
Albumin genenot expressed
Crystallin geneexpressed
(b) Lens cell
Combinatorial Control of Gene Activation
A particular combination of control elements can activatetranscription only when theappropriate activator proteinsare present
#2
Coordinately Controlled Genes
• 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
#2
Mechanisms of Post-Transcriptional Regulation
• 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
#3-6
or
RNA splicing
mRNA
PrimaryRNA transcript
Troponin T gene
Exons
DNA
Alternative RNA Splicing
different mRNA molecules are produced
from the same primary transcript,
depending on which RNA segments are treated as exons/introns
#3
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
Initiation of Translation
• 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
#5
Protein tobe degraded
Ubiquitinatedprotein
Proteasome
Protein entering aproteasome
Protein fragments(peptides)
Proteasomeand ubiquitinto be recycled
Ubiquitin
Protein Processing and Degradation• After translation, various types of protein processing,
including cleavage and the addition of chemical groups, are subject to control
proteasomes-
are giant protein complexes that bind protein molecules and degrade them
#1
#2
#3
#4
#6Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol.
The ubiquitin-tagged proteinis recognized by a proteasome,which unfolds the protein andsequesters it within a central cavity.
Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol.
#5
#6
Chromatin modification
• Genes in highly compactedchromatin are generally nottranscribed.• Histone acetylation seemsto loosen chromatin structure,enhancing transcription.
• DNA methylation generallyreduces transcription.
mRNA degradation
• Each mRNA has a characteristic life span,determined in part bysequences in the 5 and3 UTRs.
• Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors.
Bending of the DNA enables activators tocontact proteins at the promoter, initiatingtranscription.
• Coordinate regulation:Enhancer forliver-specific genes
Enhancer forlens-specific genes
Transcription
RNA processing
• Alternative RNA splicing:
Primary RNAtranscript
mRNA or
• Initiation of translation can be controlledvia regulation of initiation factors.
• Protein processing anddegradation by proteasomesare subject to regulation.
Translation
Protein processing and degradation
Chromatin modification
Transcription
RNA processing
mRNAdegradation
Translation
Protein processingand degradation
REVIEW
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
(a) Primary miRNA transcript
HairpinmiRNA
miRNA
Hydrogenbond
Dicer
miRNA-proteincomplex
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
5 3
• MicroRNAs (miRNAs)-
small single-stranded RNA molecules that can bind to mRNA• These can degrade mRNA
or block its translation
• 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
Chromatin modification
Transcription
RNA processing
mRNAdegradation
Translation
Protein processingand degradation
Chromatin modification
Translation
mRNA degradation
• miRNA or siRNA can target specificmRNAs for destruction.
• miRNA or siRNA can block the translationof specific mRNAs.
• Small or large noncoding RNAs canpromote the formation of heterochromatinin certain regions, blocking transcription.
REVIEW