Regulation of Translation

Question 1

Why do we regulate gene expression via translation ?

Answer 1

1. Rapid response and reversible
2. Regulate translation of stable mRNA’s
3. Fine tuning beyond level of transcription
   (How much protein you are making)
4. Can localize expression within one cell

Question 2

What is the function of eIF2a?

Answer 2

Brings first tRNA (initiator carrying Methionine) to the P site of small ribosomal subunit to begin translation. NEEDS GTP

Question 3

What are the two methods of regulation of translation?

Answer 3

1. Global translation control due to amino acid starvation - regulation of eIF-a
2. Targeting the CAP complex to regulate translation - regulation of eIF4E

Question 4

What is eIF2 made up of?

Answer 4

This is a heterodimer - 4 polypeptides (one subunit a = regulatory subunit CAN GET PHOSPHORYLATED

Question 5

Which method regulates eIF2-a via an eIF2a kinase?

Answer 5

Global translation control via amino acid starvation

Question 6

Explain what happens with High AA (amino acids).

Answer 6

Aminoacyl tRNA synthetases charge each tRNA with amino acid. Get Translation Initiation

Question 7

Explain what happens with LOW AA (amino acids).

Answer 7

Uncharged tRNA’s accumulate due to buildup.

eIF2 alpha kinase (GCN2) has a high affinity for uncharged tRNA’s. Uncharged tRNA’s activate GCN2 kinase.

The GCN2/uncharged tRNA then phosphorylates eIF2a at ser - 51

Question 8

What is the result of the eIF2a phosphorylation by the GCN2 kinase at ser-51?

Answer 8

Unable to recharge with GTP therefore NO TRANSLATION!

Question 9

What is the function of eIF2-a?

Answer 9

This brings in the charged net-tRNA into the P site of the ribosome to begin translation

Question 10

What happens under amino acid starvation?

Answer 10

No eIF2-a:GTP = “NO translation

But GCN4 mRNA is still translated. GCN4 ramps up amino acid’s in the cell to get translation.

Question 11

What is GCN4? And what is it’s function?

Answer 11

GCN4 is a transcription factor AND it “turns on” amino acid biosynthetic gene.

Makes more amino acids to continue translation.

Question 12

Which method regulates translation via an eIF4E focus and activates translation by insulin?

Answer 12

Targeting the CAP to regulate translation

Question 13

What is the function of 4E-BP (4E binding protein)?

Answer 13

This protein holds back the eIF4E preventing it from binding to 5’ CAP to not do its job.

Blocks translation

If we don’t want a lot of translation, then we make more 4E-BP that will hold back eIF4E to prevent translation

Question 14

What is the function of eIF4E?

Answer 14

eIF4E binds 5’ CAP - directs ribosome to mRNA for translation to occur

Question 15

How is eIF4E released to activate translation again?

Answer 15

Insulin binds to kinase receptors in the cell, triggering a kinase cascade. Activated TOR phosphorylates 4E-BP to release eIF4E to activate translation again

Question 16

How do FMRP and CYF1P1 prevent translation?

Answer 16

FMRP binds site in mRNA and attracts CYF1P1 which binds eIF4E = prevents translation initiation

Question 17

What is the function of FMRP?

Answer 17

This holds the mRNA so that translation does NOT occur unless there is s signal / synapsis

Question 18

In targeting the CAP to regulate translation, what steps are required for translation to occur and what molecule is involved in this?

Answer 18

For translation to occur, eIF4E needs to be “free” to bind the eIF4 complex - eIF4G and PABP

In response to glutamate signaling FMRP is degraded and CYR1P1 is released allowing the “free” eIF4E to bind the eIF4 complex for translation to occur.

Question 19

What is Fragile X Syndrome?

Answer 19

X-linked mutation in FMR1 gene encodes the FMRP. Where proteins are translated inappropriately!

Question 20

What is the mutation in the FMR1 gene?

Answer 20

CGG repeat in the nontranslating part

Question 21

What are some characteristics of fragile X syndrome for males?

Answer 21

1. Prominent or long ears
2. A long face
3. Delayed speech
4. Large testes
5. Hyperactivity
6. Tactile defensiveness
7. Gross motor delays
8. Autistic-like behaviors

Translating proteins inappropriately!

Question 22

What happens when we have a large number >200 CGG repeats? Full mutation?

Answer 22

Gene turns OFF and NO protein is made. Methylation and generally silencing of gene.

Question 23

What is the function of eIF3?

Answer 23

Binds to E site of 40s ribosomal subunit to block it from binding 60s subunit. Keeps them separate cause we don’t want them together at the beginning.

Question 24

What is the function of eIF2B?

Answer 24

This switches out spent GDP to get fresh GTP.

eIF2B BINDS eIF2-GDP complex in normal translation process.

Question 25

What happens if the alpha subunit of eIF2 gets phosphorylated?

Answer 25

If eIF2 alpha subunit gets phosphorylated, THEN eIF2B cannot Bind to eIF2:GDP complex to switch out GDP for GTP anymore AND TRANSLATION STOPS!

Question 26

What is eIF2 BOUND to?

Answer 26

eIF2 is bound to a GTP - NEEDS GTP

Question 27

What happens after eIF2:GTP brings in tRNAi-Met to 40s ribosomal subunit?

Answer 27

Scans for first AUG! Then 60s ribosomal subunit binds AND GTP gets hydrolyzed to GDP - eIF2:GDP.

Now this needs eIF2B to switch GDP for fresh GTP for translation cycle to continue.

Question 28

What is GCN2?

Answer 28

GCN2 is the eIF2 alpha KINASE that has a high affinity for uncharged tRNA’s and SHUTS DOWN translation. BLOCKS translation initiation

Question 29

What is the outcome of eIF2 alpha:GDP is phosphorylated due to cellular stress stimuli?

Answer 29

Decreased general transcription

AND

Increased gene specific translation

Question 30

What does a mutation in FMRP cause?

Answer 30

Causes FMR1 gene to not be able to encode a protein