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CMB2001 - Control of Eukaryotic Gene Expression > L9 - Post-transcriptional control of gene expression III > Flashcards

L9 - Post-transcriptional control of gene expression III Flashcards

1
Q

What is the ribosome?

A

The enzyme that catalyses protein synthesis

It is an RNA protein complex that mediates translation, peptide synthesis & bond formation

Mediated by tRNA – bring in new AAs

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2
Q

What is the ribosome in prokaryotes?

A

70S ribosome

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3
Q

What is the ribosome in eukaryotes?

A

80S ribosome

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4
Q

What are tRNAs?

A

Same in prokaryotes & eukaryotes

Clover leaf structure that folds to an L shape

Has 4 stems
Acceptor – amino acid joins
Anticodon – base pairs with mRNA

tRNAs need to be charged, so needs AA added to the 3’ end (acceptor stem)

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5
Q

How does a tRNA become charged?

A

Done by the enzyme tRNA synthetase (different one for each AA)

Takes the AA, adds ATP and then takes the intermediate & adds it to the end of tRNA

Results in a charged tRNA – tRNA + AA

The charged tRNA can then go into the ribosome & we get peptide bond formation

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6
Q

Peptide bond formation

A

Not a very favorable reaction

Our charged tRNA is bringing in the amino acid and docks on to the mRNA through anticodon-codon interaction

The P site is the polypeptide chain site, the tRNA is covalently lined on the growing peptide chain, it is always connected until translation is complete

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7
Q

How does translation elongation occur?

A

We transfer the AA on the to the peptide chain & the other tRNA is detached

Then a ratcheting mechanism occurs & the mRNA is moved through the ribosome

This is done by elongation factor G
This comes in hydrolyses GTP & shunts the mRNA & tRNA one step through the ribosome

The tRNA that was in the P site moves to the E site, & A site to P site

This then repeats

Then the tRNA that is in the E site leaves, & reattaches an AA so it can be reused

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8
Q

2 key components of eukaryotic mRNAs

A

m7G cap structure at the 5’

PolyA tail

Without the cap cant initiate translation, & polyA tail is required for efficient reinitiating of translation

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9
Q

What does eukaryotic translation initiation depend on?

A

The 5’ cap of the RNA

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10
Q

How dies the 5’ cap initiate eukaryotic translation?

A

Small subunit binds to cap, and it moves along until it finds the start codon, AUG

Once the small subunit is it at the start site, the large subunit comes in and forms the complete ribosome around the start codon

Without cap recognition translation doesn’t occur in most RNAs

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11
Q

What is the Kozak consensus?

A

The optimal translation initiation sequence

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12
Q

Translation initiation – circularising the mRNA

A

PolyA tail bound by polyA binding protein (PABP)

Cap is bound by eIF4F complex, made up on E, G & A

Get circularization as eIF4G interacts with PABP

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13
Q

What is circularising the mRNA important?

A

Circular structure important as ribosomes thought to come in, initiate translation, translate through open reading frame, and can then easily be position back to start site, so can easily repeat

Circularization also important for stability of RNA

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14
Q

WHat do the e & the I stand for in the eIF4F complex?

A

e = eukaryotic

I = initiation F-factor

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15
Q

Whats the half-life of eukaryotic ribosomes?

A

70 hours

SO does lots of rounds of translation

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16
Q

What is the ribosome recycled to?

A

40s and 60s subunits

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17
Q

How is translation initiated in eukaryotes?

A

1) The small subunit interacts with the EIF2 ternary complex to form a 40s pre initiation complex
2) The complex then attaches to our circular mRNA to form the initial complex
3) Ribosome scans from the cap along, from the 5’ to 3’, looking for the start codon
4) When it gets to the AUG, we get hydrolysis of GTP by EIF2, so we start to loose EIF2 and we form the 40s initiation complex
5) This complex is ready to bind the large subunit
6) The large subunit comes in with EIF5b which is another GTPase
7) We get release of some factors and we form our complete ribosome
8) Then we have the complex formed, we get hydrolysis of the GTP, then we have the 80s initiation complex which is capable of translation

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18
Q

What is the EIF2 ternary complex made of?

A

Made of EIF2 which is a GTPase, and Met-tRNA (methione initiator tRNA)

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19
Q

What is the importance of the 40s pre-imitation complex?

A

This complex is formed, so before the mRNA is even bound, we have a tRNA in our small ribosomal subunit

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20
Q

Where is the ribosome located in the initial complex?

A

Near the cap structure

It is recruited through the cap

21
Q

Why is the initial complex not ready for translation?

A

We only have 40s subunit, and its not at the start codon, so it needs to move

22
Q

Translation elongation in eukaryotes

A

Elongation is relatively simple compared to initation

1) Bringing in new tRNAs with eEF1A
2) Peptide bond formation
3) Translocation - mRNA moves along
4) More peptide bond formation
5) Another tRNA coming in
6) When we get to the end of mRNA, we get to the termination codon, there’s no tRNA that binds termination codon, so it remain free

23
Q

What happens once the termination codon is reached?

A

Its then bound by a protein ERF1 – eukaryotic release factor 1

24
Q

What does ERF1 do in translation termination?

A

This is the first protein that binds to release translation

Comes in binds at a site & triggers release of peptide
Triggers release of the two subunits through another factor ERF3

Then we get the start of the initiation factor again

All components are re used and recycled

25
Q

What is elF2B?

A

This is a tRNA that is involved in making the initial complex with the initiator tRNA

This is the main mechanism where you would control translation

eIF2B subunit of eIF2

26
Q

What does elF2B control?

A

eIF2B level governs level of active eIF2-GTP and thus overall initiation rate, so its controlling initiation

Activated by insulin

27
Q

When is elF2B down-regulated?

A

In response to stresses such as viral infection & amino-acid deprivation

Also in diabetic muscle

28
Q

Regulation of translation initiation

A

Whilst we have active translation everything is fine, but when we have stress, we get inactivation of eIF2 by phosphorylation and this blocks translation

So, we control the amount of initiator tRNA and this complex by phosphorylation

So, when phosphorylation happens, we can switch to the stress specific mRNA translation e.g. heat shock

29
Q

What does elF2 do to initiate translation?

A

eIF2 comes in with initiator tRNA in the complex that starts on the mRNA, so it brings in the first tRNA, it is only needed for initiation

30
Q

Phosphorylation of elF2 under stress

A

eIF2 is controlled under stress, its phosphorylation is blocked by essential amino acids

All these stresses cause phosphorylation of eIF2 which blocks eIFB which blocks protein synthesis

31
Q

Regulation of iron metabolism

A

The levels of Fe in the cell regulate the expression of Fe-storage/transport proteins

Fe found in heme and iron-sulfur clusters

32
Q

Why does iron need to be regulated in the body?

A

Free irons not good for us - toxic

Not enough iron is toxic as we need iron containing proteins

Iron in the cell needs to be bound, it needs to be stored, exported or utilised to make iron protein complexes

33
Q

What proteins get rid of iron from the cell?

A

Ferroportin

34
Q

What proteins store iron in the cell?

A

Ferritin

35
Q

Regions in mRNAs that regulate translation?

A

Have 2 UTR, one 5’ and one 3’ in every mRNA

Elements in both the 5’ and 3’ UTRs can influence translation

36
Q

What are UTRs?

A

Untranslated regions

Between cap and start and stop and polyA

They are encoded in the genome

Vary in length

37
Q

2 classes of proteins involved in iron level regulation

A

Factors involved in storage
Factors involved in transport

Need to be regulated differently as don’t need them both at the same time

38
Q

What happens when we have low iron?

A

We want transport but not storage

Under low iron conditions all of our storage proteins are translationally repressed, so we’re not translating storage proteins, but we are translating transport proteins

39
Q

What happens when we have high iron?

A

We want storage but not transport

Under high iron conditions we get translational activation of storage proteins, and mRNA turnover and block in translation of transport proteins

40
Q

What controls whether iron is stored or transported?

A

Its switched through a protein called Iron response protein 1 and 2 – IRP1/2

41
Q

How is IRP1/2 involved in translational repression in storage when theres low iron?

A

It binds to a little sequence in the 5’ UTR of the mRNAs

This protein binds the mRNAs and blocks translation, probably blocks the ribosome scanning from cap to start codon

42
Q

How is IRP1/2 involved in mRNA stabilisation of transport factors when theres low iron?

A

The mRNAs are bound by same IRP, but these are bound in the 3’ UTR, which cause the mRNA to be stabilized, without these proteins binding the mRNAs are unstable

43
Q

What happens to IRP1/2 when we have high iron?

A

IRP1 is bound to an iron sulphur cluster

IRP2 is degraded

44
Q

Why is IRP1 being bound to an iron sulphur cluster important for translational activation of storage protein genes?

A

The protein is blocked & can’t bind mRNA

Cannot bind the 5’ site of storage protein genes

mRNA are translated with no issues as ribosome can scan RNA and translate

45
Q

Why is IRP1 being bound to an iron sulphur cluster important for mRNA degradation of transport protein genes?

A

IRP1 cannot bind to the 3’ site so mRNA is unstable so is degraded

46
Q

What are IREs?

A

Iron response elements

Got stem structures that slightly interrupted by a C then a loop

Nucleotides in red are very conserved and are important for binding the protein

47
Q

What are the 2 forms of IRP

A

With or without the iron sulphur cluster

Without it, it binds the IRE in the mRNA

With the cluster its an enzyme called c-aconitase

48
Q

What does IRP stand for?

A

Iron regulatory protein

49
Q

What does c-aconitase do?

A

Interconverts citrate and isocitrate and it has an iron sulphur cluster in its catalytic core

This enzyme cannot function without an iron sulphur cluster, but it can go and regulate iron levels in the cell by going and regulating the translation of iron storage and transport proteins

So can switch from using iron to making the cell use more or less iron

CMB2001 - Control of Eukaryotic Gene Expression (10 decks)

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