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


Refresher: Translation


Read the chapter 7 in your text book, Essential Cell Biology (3rd Edition)

Questions to [email protected], Rm 3114


Eukaryotes and prokaryotes have different mRNA transport for translation


Eukaryotes and prokaryotes have different sequences upstream of ATG (start codon)

Kozak sequence

Is not ribosome binding site

RBS is 5’ cap of mRNA

Essential for translation initiation

Shine Dalgarno sequence

Is the ribosome binding site

E.coli

Human


Eukaryotes and prokaryotes have different mRNA transport for translation


A molecule called transfer RNA (tRNA) recognizes the messenger RNA (mRNA) codon and adds the corresponding amino acid


Amino acid specific synthetase enzymes “charge” the tRNA with the correspondingamino acid.


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


Ribosome produces protein in a 4 step process

LARGE

SMALL


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


How does this process all end?

Ends with binding of the release factor binding to stop codon (UAG)


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


Control of Gene Expression, Manipulating Genes

Tobias Schoep


Read the chapter 8 in your text book, Essential Cell Biology (3rd Edition)

Questions to [email protected], Rm 3114


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)


Permanent changes to gene expression:

Propagation of condensed chromatin structure

Positive feed back loop (eukaryotes)


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

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


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 11/03/11ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

27

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


lactose

active repressor (LacI)

inactive repressor

cAMP - ↓ glucose

inactive activator

active activator

Components:


active repressor

active activator

Activator and Repressor binding sites:


↓cAMP

↓cAMP

cAMP - ↓ glucose

lactose

active repressor

active activator


↑cAMP

Lac operon based expression systems are commonly used to produce proteins


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


Other mechanisms of regulating gene expression


Other mechanisms of regulating gene expression

Post-transcriptional controls operate after RNA polymerase has produced mRNA

Conformational change in RNA can regulate gene expression


RNA switch affecting transcription (Riboswitch)


Riboswitch Antisense RNA

RNA switches affecting translation


RNA switches affecting RNA processing and degradation

microRNA (miRNA) regulates up to 1/3 of protein coding genes

RNA-induced silencing complex


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


SUMMARY

ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11ChE 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


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


Based on bacterial antibiotic resistance system

Small amount of tetracycline turns on genes coding pumps to export tetracycline

Components:

tetR tetO tetA

TetRTetR

P P

tetR tetO tetA

TetRTetR

P P

tetracycline


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)


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

tetRP

TRE GOIP

VP16 tTA

tTA activation

TRE GOIP

tTA

doxycycline


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)


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

rtetRP

TRE GOIP

VP16 rtTA

rtTA activation

TRE GOIP

tTA

doxycycline


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


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


Used Tet (OFF) system to turn on MYC oncogene expression when doxycycline removed from mouse drinking water

TetRP VP16 tTAEnhancer

TRE MYCP

tTA activation

TRE MYCP

tTA

doxycycline in drinking water

Remove doxycycline from drinking water

Liver specific

basal myc

↑ myc


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

Kim et al. (2011) Generation of Transgenic Dogs that Conditionally Express Green Fluorescent Protein, Genesis, 49:472–478

TRE eGFPP

rtTA activation

doxycycline


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

ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

Documents

Transcript of ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11