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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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