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Chapter 16
Control of Gene Expression(Part 2)
Gene Regulation
• Unicellular flexibility– Genes turned on and off in response to
environment
• Multicellular specialization– Genes for one cell type are not expressed in
other cell types
Figure 16.1
Levels of Gene Regulation
• Gene Structure• Transcription• mRNA processing• Regulation of mRNA stability• Translation• Post translational protein modification
Genes vs. Regulatory Elements
• Structural genes:– Metabolism, structure, biosynthesis
• Regulatory genes:– Affect transcription or translation– DNA binding proteins
• Regulatory elements:– Not transcribed– Affect gene expression
Bacterial Gene Regulation
• Functionally related genes often clustered• Can be transcribed together on same
mRNA
•• OperonOperon:: Group of bacterial structural genes that are transcribed together.– includes promoters and regulatory elements
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Modes of Transcriptional Control
•• NegativeNegative– Regulatory protein acts as repressor– Bind to DNA and inhibits transcription
•• PositivePositive– Regulatory protein acts as activator– Binds to DNA and stimulates transcription
2 Classes of Operon
•• InducibleInducible– Transcription is normally OFFOFF– Modulator turns transcription ON
•• RepressibleRepressible– Transcription is normally ONON– Modulator turns transcription OFF
An ExampleAn Example: The lac Operon of E. coli
• Involved in lactose metabolism in E. coli•• Lactose:Lactose:
– Disaccharide– Doesn’t diffuse across membrane easily
•• Enzymes:Enzymes:– Β-Galactosidase– Permease– Transacetylase
Figure 16.6Figure 16.6
F’ Cells
• Cells containing an F plasmid with some bacterial genes.
Figure 8.16
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Partial Diploids
• Conjugation between an F’ Cell and an F-cell can result in cells with 2 copies of some genes
• These are called Partial Diploids or merozygotes
Partial Diploids will come in really handy for
studying gene expression! (Chapter 16)
laclac Mutations
•• Partial DiploidPartial Diploid strains of E. coli:– 2 copies of lac operon– Bacterial chromosome– Plasmid
•• CisCis actingacting mutations:– Control expression of genes on the same piece of
DNA only
•• Trans actingTrans acting mutations:– Control expression of genes on other DNA molecules
Genotypes of Partial Diploids
• Bacterial Chromosome / / Plasmid
• Examples:– lacZ- lacY+ / lacZ+ lacY-
– Structural mutation of lacZ gene on bacterial chromosome
– Structural mutation of lacY gene on plasmid
Figure 16.10Figure 16.10lacI+ lacZ- / lacI- lacZ+
Plasmid
Plasmid
Chromosome
Chromosome
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Types of Mutations I
• Structural gene Mutations– Affect structure of enzymes, not regulation– The wild type is Trans DominantTrans Dominant
• Regulator gene Mutations–– Constitutive:Constitutive: lac enzymes produced
constantly (in regular E. coli)– In partial diploids, lacIlacI++ is Trans DominantTrans Dominant
Figure 16.11Figure 16.11
lacIs encodes a superrepressor
lacIs lacZ+ / lacI+ lacZ+
Types of Mutations II
• Operator mutations– lacOc indicates a mutation in the DNA
sequence of the operator– Repressor cannot bind to operator– lacOc is ciscis dominant dominant and constitutiveconstitutive
Figure 16.12Figure 16.12
Constitutive!Constitutive!
Figure 16.11Figure 16.11
Constitutive!Constitutive!
Figure 16.11Figure 16.11
CisCis acting!acting!
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Figure 16.11Figure 16.11
CisCis acting!acting!
Types of Mutations III
• Promoter mutations– lacP- indicates a mutation in the DNA
sequence of the promoter– RNA polymerase cannot bind to promoter– lacP- is ciscis dominantdominant
lacI+ lacP- lacOc lacZ+ lacY- / lacI- lacP+ lacO+ lacZ- lacY+
• What is the ENZYMATIC ACTIVITYENZYMATIC ACTIVITY?
•• Lactose AbsentLactose Absent Lactose PresentLactose Present• B-Gal Permease B-Gal Permease• ? ? ? ?
• Use “-” for no activity and “+” for activity
lacI+ lacP- lacOc lacZ+ lacY- / lacI- lacP+ lacO+ lacZ- lacY+
• What is the ENZYMATIC ACTIVITYENZYMATIC ACTIVITY?
•• Lactose AbsentLactose Absent Lactose PresentLactose Present• B-Gal Permease B-Gal PermeaseA) + - + +B) - - + +C) - - - +D) + + + -
Figure 16.9Figure 16.9lacI+ lacZ- / lacI- lacZ+
Plasmid
Plasmid
Chromosome
Chromosome
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Catabolite Repression
•• GlucoseGlucose is the preferred food source for E. coli
• When glucose is available:– Genes for metabolism of other sugars are
repressed–– CataboliteCatabolite RepressionRepression
CAP and cAMP•• CataboliteCatabolite AcitvatorAcitvator ProteinProtein
– Binds to DNA upstream of lac promoter– RNA polymerase won’t bind efficiently to lac
promoter unless CAP is first bound to DNA•• Cyclic AMPCyclic AMP (adenosine-3’,5’-cyclic
monophosphate)–– CAPCAP can’t bind to DNA without cAMPcAMP– Concentration of cAMP inversely proportional
to glucose concentration
Figure 16.12Figure 16.12
This is POSITIVE CONTROLPOSITIVE CONTROLbecause CAPCAP is an ACTIVATORACTIVATOR
trp Operon
• Controls biosynthesis of tryptophan•• Negative repressibleNegative repressible operon
Figure 16.14Figure 16.14
Attenuation
• Another form of transcriptional control for the trp operon.
• Transcription is initiated but terminates prematurely.
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Figure 16.14Figure 16.14 Figure 16.14Figure 16.14
Look Familiar?Look Familiar?
Rho-independent Termination Rho-independent Termination
1) Two inverted repeats in the DNA sequence are transcribed
2) A string of ~6 Adenines follows the second inverted repeat
3) The inverted repeats form a hairpin structure pausing the polymerase
4) The A-U bonds break and the RNA molecule separates from the template
Figure 16.14Figure 16.14
Check out the online
animation for the lac operonand attenuation
Antisense RNA• RNA regulator of gene expression•• AntisenseAntisense RNARNA
– Small RNA molecules complementary to certain sequences on mRNAs.
– Inhibit translation• Example: ompF gene of E. coli
– Important in cellular osmoregulation– Increased osmolarity turns on micF– micF produces Antisense RNA
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Figure 16.17Figure 16.17 Figure 16.17Figure 16.17
Ribosome cannot bindRibosome cannot bind
Eukaryotic Gene RegulationEukaryotic Gene Regulation
Eukaryotic Gene Regulation
• No operons in Eukaryotes• Chromatin affects gene expression• Activators are more common• Many mechanisms at many levels
• Gene Structure• Transcription• mRNA processing• Regulation of mRNA stability• Translation• Post translational protein modification
Eukaryotic Gene Regulation Gene Regulation:Gene Regulation: Gene Structure (Chromatin)
•• DNAaseIDNAaseI HypersensitivityHypersensitivity– DNAase I digests DNA– Doesn’t work when DNA tightly bound to
histones• Transcriptionally active genes
– DNAaseI hypersensitive sites• Regions near transcriptionally active genes where
DNA configuration is more open• DNA binding proteins?
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Gene Regulation:Gene Regulation: Gene Structure (Chromatin) cont.
•• HistoneHistone acetylationacetylation– Facilitates transcription
•• DNA DNA methylationmethylation– Cytosine bases methylated– Associated with transcription repression–– CpGCpG islandsislands:
…GC……CG…
Gene Regulation:Gene Regulation: Transcriptional Control
• Transcriptional activators– Stabilize basal transcription apparatus basal transcription apparatus
(BTA)(BTA)– Often interact with BTA through coactivatorscoactivators– Stimulate transcription
• Repressors– May bind to regulatory promoter– May bind to silencerssilencers
ENHANCERS AND INSULATORS
• Enhancers affect transcription at distant promoters– Alpha chain of Tcell receptor: enhancer is
69,000 bp downstream of promoter– Enhancers can stimulate any promoter in its
vicinity•• InsulatorsInsulators (boundary elements) limit the
effect of enhancers
Figure 16.23Figure 16.23
RESPONSE ELEMENTS•• Response elementsResponse elements
– DNA regulatory elements which are bound by transcriptional activator proteins.
• Example: Metallothionein– Response elements to heavy metals
• Eukaryotic Genes may be activated by several different response elements
Multiple Response Elements (MREs)
allow the same gene to be activated by different stimuli.
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Response elements to a particular stimulus can be associated with multiple genes, allowing a single
stimulus to activate multiple genes.
Gene Regulation:Gene Regulation: Messenger RNA Processing
•• Alternative SplicingAlternative Splicing:–– SR Proteins: SR Proteins: often regulate splicing
– Example: T-antigen gene of mammalian virus SV40
• Splicing Factor 2 (SF2SF2) is a type of SR ProteinSR Protein
– Another Example: Sex determination in Drosophila.
Gene Regulation:Gene Regulation: RNA Stability
• Stability of mRNA affected by:– 5’ Cap– Poly (A) tail– 5’ UTR– Coding region– 3’ UTR
Variation in Variation in mRNA StabilitymRNA Stability
Variation in Protein Variation in Protein ProductionProduction
Gene Regulation:Gene Regulation: Translation and Posttranslational Control
• Availability of Translational ApparatusTranslational Apparatus:– Ribosomes, aminoacyl tRNAs, initiation factors,
elongation factors.– Less available: slower translation.
• Proteins binding to 5’ UTR
• Posttranslational modification– Trimming, acetylation, addition of phosphates,
carboxyl groups, etc.
RNA Interference (RNA Silencing)
• Double stranded RNA initiates a cascade that degrades complementary mRNA.
• May have evolved as a defense against double stranded RNA viruses.
• Very handy for artificially regulating gene expression– Model organisms– Genetically engineered organisms
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