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ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Control of Gene Expression, Manipulating Genes
Tobias Schoep
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Refresher: Translation
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Read the chapter 7 in your text book, Essential Cell Biology (3rd Edition)
Questions to [email protected], Rm 3114
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Eukaryotes and prokaryotes have different mRNA transport for translation
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Eukaryotes and prokaryotes have different sequences upstream of ATG (start codon)
Kozak sequence
Shine Dalgarno sequence
Is not ribosome binding site
Is the ribosome binding site
RBS is 5’ cap of mRNA
E.coli
Essential for translation initiation
Human
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Eukaryotes and prokaryotes have different mRNA transport for translation
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
A molecule called transfer RNA (tRNA) recognizes the messenger RNA (mRNA) codon
and adds the corresponding amino acid
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Amino acid specific synthetase enzymes “charge” the tRNA with the corresponding
amino acid.
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Amino acid specific synthetase enzymes “charge” the tRNA with the corresponding
amino acid.
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Ribosome de-codes (translates) mRNA into a new protein using charged tRNA
The ribosome is a composed of a large and small subunit, proteins and structural RNA
called ribosomal RNA (rRNA)
Ribosome moves along the mRNA, captures complementary tRNA, hold these in
position, and covalently links amino acids together.
Aminoacyl-tRNA
Peptidyl-tRNA
Exit
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Ribosome produces protein in a 4 step process
SMALL
LARGE
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
How does this process all start?
Starts with binding of the initiator tRNA, which is always bound to Met, and binds START
codon (AUG)
Eukaryote:
• 5’ cap tells ribosome where to start searching for START
• single, spliced, mRNA transcribed for each protein
Prokaryotes:
• ribosome recognizes ribosome binding sequence (RBS)– Shine dalgarno sequence
• ribosome can recognize multiple START sites in one mRNA molecule as long as RBS is
present
• one mRNA protein can encode several different proteins
Eukaryote
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
How does this process all end?
Ends with binding of the release factor binding to stop codon (UAG)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Ribosome is relatively conserved between eukaryotes and prokaryotes
Eukaryote ribosome adds about 2 aa / s, prokaryote ribosome adds about 20 aa / s
Multiple ribosomes can be bound to one mRNA - polyribosomes
Initiation, translation and termination
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Control of Gene Expression, Manipulating Genes
Tobias Schoep
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Read the chapter 8 in your text book, Essential Cell Biology (3rd Edition)
Questions to [email protected], Rm 3114
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Why is controlling gene expression important?
All cell have the same DNA
Eukaryotes: 25 000 genes – 5000 to 15000 expressed
Prokaryotes: 4300 genes
Some housekeeping genes are always produced
How do we get different cells with different activities?
Permanent and transient changes to gene expression such as
1.
Cellular differentiation (permanent)
2.
Cellular responses to stimuli (transient)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Permanent changes to gene expression:
Propagation of condensed chromatin structure
Positive feed back loop (eukaryotes)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Permanent changes to gene expression:
DNA methylation (eukaryotes and prokaryotes) influences how regulatory proteins bind
to DNA
Pattern changed in fetal development affects cellular differentiation
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Transient changes to gene expression:
Changes to environmental stimuli
Primary control is at the level of transcription ie. how much mRNA is produced
Differences between eukaryotes and prokaryotes
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Transient changes to gene expression:
Pokaryotes have operons: multiple genes produce one mRNA transcript and are regulated
together.
Eukaryotes generally have individual regulated genes but regulation is more complex
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Repressor and Activators:
Repressors turn off genes, activators turn them on
Activator and repressor control of the Lac Operon in bacteria (prokaryote)
Controls genes for using different carbon sources
E.coli use glucose before lactose
Enzymes for glucose use are made constantly
Enzymes for lactose use are made at very low levels in the presence of glucose
When glucose runs out and lactose is present Lac Operon is “turned on”
Lac operon encodes genes for lactose transport (lacY) and conversion (lacZ) to glucose
and galactose, and lacA
27
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Components:
lactose
cAMP - ↓ glucose
active repressor (LacI)
inactive activator
inactive repressor
active activator
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Activator and Repressor binding sites:
active activator
active repressor
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
lactose
cAMP - ↓ glucose
active activator
active repressor
↓cAMP
↓cAMP
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
↑cAMP
Lac operon based expression systems are commonly used to produce proteins
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Enhancers:
Eukaryotes use activators and repressors as well.
Activators bind to regions called enhancers
Enhancers can bind to affect transcription from great distances
In general:
Activation depends on presence of multiple factors with act in combination
Genes are often controlled by multiple sets of regulators ie. same gene can be expressed
in response to different sets of signals
However, a change in one transcription factor can cause profound changes in cell function
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Other mechanisms of regulating gene expression
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Other mechanisms of regulating gene expression
Post-transcriptional controls operate after RNA polymerase has produced mRNA
Conformational change in RNA can regulate gene expression
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
RNA switch affecting transcription (Riboswitch)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
RNA switches affecting translation
Riboswitch
Antisense RNA
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
RNA switches affecting RNA processing and degradation
microRNA (miRNA) regulates up to 1/3 of protein coding genes
RNA-induced silencing complex
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
RNA switches affecting RNA processing and
degradation
RNA interference (iRNA) detects foreign double
stranded RNA (some viruses)
Triggers cleavage to form small interfering RNAs
(siRNA)
Bind RISC and target foreign RNA for degradation
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
SUMMARY
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
What is the relevance of all this information for genetic and chemical engineers?
Using expression systems from prokaryotes and eukaryotes we can produce proteins and
other metabolites when and where we want them.
Different systems can be used for
prokaryotic cell: eg. ara/lac/trp expression systems
eukaryotic cell: eg. siRNA to study gene function in mammalian cells
animals: eg. Tet ON/OFF system to study gene function in animals
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Inducible gene expression system in animals
Use of Tet ON/OFF system to study gene function in animals
Turn on and off genes in animals
Tetraycycline (antibiotic) responsive system
Tetracycline approve human therapy
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Based on bacterial antibiotic resistance system
Small amount of tetracycline turns on genes coding pumps to export tetracycline
Components:
TetR
tetR
TetR
P
P
tetO
tetA
tetracycline
TetR
TetR
tetR
P
P
tetO
tetA
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (OFF) system
Altered for use in eukaryotes
Turn the TetR repressor into tetraycycline transactivator (tTA)
Fused VP16 to C-terminus of TetR
VP16 is from Herpes simplex virus and recruits RNA polymerase to site of promoter
Seven copies of TetO upstream of promoter of gene of interest (GOI).
Fusion of 7 operators is called the tetracycline response element (TRE)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (OFF) system
In presence of tetracycline or doxycycline (tetracycline derivative), tTA (tetracycline
transactivator) does not bind TetO in tetracycline response element (TRE), and there is
reduced expression
P
tetR
VP16
tTA
activation
TRE
P
tTA
GOI
doxycycline
tTA
TRE
P
GOI
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (ON) system
Reverse system. Mutated TetR (rTetR) binds tetO in TRE in presence of tetracycline
Modified TetR fused to VP16 is the tetraycycline responsive transactivator (rtTA)
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (ON) system
In presence of tetracycline or doxycycline (tetracycline derivative), rtTA (tetracycline
responsive transactivator) does bind TetO in tetracycline reseponse element (TRE), and
there is increased expression
P
rtetR
VP16
rtTA
doxycycline
rtTA
TRE
activation
P
GOI
P
GOI
tTA
TRE
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
More advanced tet based systems
Tet-On Advanced and Tet-On 3G: Increased sensitivity to doxycycline and lower basal
expression.
All very interesting, but what are the tet systems good for
1. Expression of reporter systems in animals such that localization of protein expression
can be examined
2. Deletion of specific cells in animals such that cell function and regeneration can be
examined
3. Construction of models that mimic human diseases
4. Regulatable models of tumorgenesis
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (OFF): Regulatable models of tumorgenesis
Study of hepatocelluar carcinoma (liver cancer)
Examined role of MYC oncogene, gene with potential to cause cancer, at different stages
of mouse development
Beer et al. (2004) Developmental Context Determines Latency of MYC-Induce Tumors, Plos Biology,
2(11):1785-1798
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Used Tet (OFF) system to turn on MYC oncogene expression when doxycycline removed
from mouse drinking water
Liver specific
Enhancer
P
TetR
VP16
tTA
doxycycline in drinking water
tTA
TRE
P
MYC
basal myc
Remove doxycycline from drinking water
tTA
TRE
activation
P
MYC
↑ myc
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (ON):
Study of transgenic cloned dogs
Generation of transgenic dogs that conditionally express green fluorescent protein
Transfer of DNA to an egg cell from which nuclear DNA had been removed
doxycycline
rtTA
TRE
activation
P
eGFP
ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11
Tet (ON):
Addition of doxycycline causes induces eGFP production
Kim et al. (2011) Generation of Transgenic Dogs that Conditionally Express Green Fluorescent Protein , Genesis, 49:472–478