Download Chapter 16 Gene Regulation Levels of Gene Regulation Bacterial

Gene Regulation
• Unicellular flexibility
– Genes turned on and off in response to
environment
Chapter 16
Control of Gene Expression
(Part 2)
• Multicellular specialization
– Genes for one cell type are not expressed in
other cell types
Levels of Gene Regulation
•
•
•
•
•
•
Gene Structure
Transcription
mRNA processing
Regulation of mRNA stability
Translation
Post translational protein modification
Figure 16.1
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
• Operon:
Operon: Group of bacterial structural
genes that are transcribed together.
– includes promoters and regulatory elements
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Modes of Transcriptional Control
• Negative
– Regulatory protein acts as repressor
– Bind to DNA and inhibits transcription
• Positive
– Regulatory protein acts as activator
– Binds to DNA and stimulates transcription
2 Classes of Operon
• Inducible
– Transcription is normally OFF
– Modulator turns transcription ON
• Repressible
– Transcription is normally ON
– Modulator turns transcription OFF
Figure 16.6
An Example:
Example The lac Operon of
E. coli
• Involved in lactose metabolism in E. coli
• Lactose:
– Disaccharide
– Doesn’t diffuse across membrane easily
• Enzymes:
– Β-Galactosidase
– Permease
– Transacetylase
F’ Cells
• Cells containing an F plasmid with some
bacterial genes.
Figure 8.16
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Partial Diploids
Partial Diploids will
come in really handy for
studying gene
expression!
(Chapter 16)
• Conjugation between an F’ Cell and an Fcell can result in cells with 2 copies of
some genes
• These are called Partial Diploids or
merozygotes
Genotypes of Partial Diploids
lac Mutations
• Partial Diploid strains of E. coli:
• Bacterial Chromosome / Plasmid
– 2 copies of lac operon
– Bacterial chromosome
– Plasmid
• Examples:
• Cis acting mutations:
– Control expression of genes on the same piece of
DNA only
• Trans acting mutations:
– lacZ- lacY+ / lacZ+ lacY– Structural mutation of lacZ gene on bacterial
chromosome
– Structural mutation of lacY gene on plasmid
– Control expression of genes on other DNA molecules
lacI+ lacZ- / lacI- lacZ+
Figure 16.10
Chromosome
Plasmid
Chromosome
Plasmid
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Figure 16.11
Types of Mutations I
lacIs lacZ+ / lacI+ lacZ+
• Structural gene Mutations
– Affect structure of enzymes, not regulation
– The wild type is Trans Dominant
• Regulator gene Mutations
– Constitutive: lac enzymes produced
constantly (in regular E. coli)
– In partial diploids, lacI+ is Trans Dominant
lacIs encodes a superrepressor
Figure 16.12
Types of Mutations II
Constitutive!
• Operator mutations
–
lacOc
indicates a mutation in the DNA
sequence of the operator
– Repressor cannot bind to operator
– lacOc is cis dominant and constitutive
Figure 16.11
Figure 16.11
Constitutive!
Cis acting!
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Figure 16.11
Types of Mutations III
• Promoter mutations
Cis acting!
– lacP- indicates a mutation in the DNA
sequence of the promoter
– RNA polymerase cannot bind to promoter
– lacP- is cis dominant
lacI+ lacP- lacOc lacZ+ lacY- /
lacI- lacP+ lacO+ lacZ- lacY+
• What is the ENZYMATIC ACTIVITY?
ACTIVITY
• Lactose Absent
Lactose Present
• B-Gal
•
?
B-Gal
?
Permease
?
Permease
?
• Use “-” for no activity and “+” for activity
lacI+ lacP- lacOc lacZ+ lacY- /
lacI- lacP+ lacO+ lacZ- lacY+
•
What is the ENZYMATIC ACTIVITY?
ACTIVITY
•
Lactose Absent
Lactose Present
•
A)
B)
C)
D)
B-Gal Permease
+
+
+
B-Gal
+
+
+
Permease
+
+
+
-
lacI+ lacZ- / lacI- lacZ+
Figure 16.9
Chromosome
Plasmid
Chromosome
Plasmid
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Catabolite Repression
CAP and cAMP
• Glucose is the preferred food source for
E. coli
• Catabolite Acitvator Protein
• When glucose is available:
• Cyclic AMP (adenosine-3’,5’-cyclic
monophosphate)
– Genes for metabolism of other sugars are
repressed
– Catabolite Repression
– Binds to DNA upstream of lac promoter
– RNA polymerase won’t bind efficiently to lac
promoter unless CAP is first bound to DNA
– CAP can’t bind to DNA without cAMP
– Concentration of cAMP inversely proportional
to glucose concentration
Figure 16.12
trp Operon
• Controls biosynthesis of tryptophan
• Negative repressible operon
This is POSITIVE CONTROL
because CAP is an ACTIVATOR
Figure 16.14
Attenuation
• Another form of transcriptional control for
the trp operon.
• Transcription is initiated but terminates
prematurely.
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Figure 16.14
Figure 16.14
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 16.14
Antisense RNA
• RNA regulator of gene expression
• Antisense RNA
Check out the
online
animation for
the lac operon
and attenuation
– 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 16.17
Figure 16.17
Ribosome cannot bind
Eukaryotic Gene Regulation
•
•
•
•
No operons in Eukaryotes
Chromatin affects gene expression
Activators are more common
Many mechanisms at many levels
Eukaryotic Gene Regulation
Eukaryotic Gene Regulation
•
•
•
•
•
•
Gene Structure
Transcription
mRNA processing
Regulation of mRNA stability
Translation
Post translational protein modification
Gene Regulation: Gene Structure
(Chromatin)
• DNAaseI Hypersensitivity
– 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 Structure
(Chromatin) cont.
• Histone acetylation
– Facilitates transcription
• DNA methylation
– Cytosine bases methylated
– Associated with transcription repression
– CpG islands:
islands
…GC…
…CG…
Gene Regulation: Transcriptional
Control
• Transcriptional activators
– Stabilize basal transcription apparatus
(BTA)
– Often interact with BTA through coactivators
– Stimulate transcription
• Repressors
– May bind to regulatory promoter
– May bind to silencers
Figure 16.23
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
• Insulators (boundary elements) limit the
effect of enhancers
RESPONSE ELEMENTS
• Response 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: Messenger
RNA Processing
• Alternative Splicing:
Splicing
– SR Proteins: often regulate splicing
– Example: T-antigen gene of mammalian virus
SV40
• Splicing Factor 2 (SF2
SF2) is a type of SR Protein
– Another Example: Sex determination in
Drosophila.
Gene Regulation: Translation and
Posttranslational Control
• Availability of Translational Apparatus:
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.
Gene Regulation: RNA Stability
Variation in
mRNA Stability
Variation in Protein
Production
• Stability of mRNA affected by:
– 5’ Cap
– Poly (A) tail
– 5’ UTR
– Coding region
– 3’ UTR
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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