Lecture 24 - Translation Continued

Question 1

What was the VERY DRAMATIC EXPERIMENT that bob refered to ie what was the dramatic conclusion

Answer 1

The growth of the fruitfly larvae can be markedly enhanced by overexpressing initiator tRNA (met-tRNA^met) - - - - -therefore translation initiation in rate limiting for fruitfly growth at this stage

Question 2

Describe the very dramatic experiment

Answer 2

Exp: Overexpress diff types of tRNA in developing fruit fly larave
Results: overexpressing initiator tRNA caused a big increase in the size of pupae developed from that larvae (The result was bigger than the initial overesxpression)
This was only seen with initiator tRNAs not others

Question 3

What are the 2 key points of control in euk translation initiation

Answer 3

assembly of cap binding complex

eIF2 - ie the deliverance of tRNA to the PIC

Question 4

What eIF2 does (very brief because it all in previous lecture)

Answer 4

delivers initiator tRNA to 40s subunit as part of ternary complex (with GTP)
Has 3 subunits to recognise the tRNA’s unique structure
Has GTPase activity
Once start codon recognised, eIF2 hydrolyes GTP and the process is now irreversible

Question 5

How is eIF2 recycled?

Answer 5

eIF2 attracts eIF2B
eiF2B binds preferentially to eiF2 when it is GDP bound
This causes eIF2 to revert to it’s GTP bound state and be once again active

Question 6

What condition prevent the recycling on eIF2 and therefore surpress ttranslation?

Answer 6

Stress.

Question 7

What impact does stress have on eIF2

Answer 7

eIF2 phosphorylated by eIF2 kinases, sequestering eiF2B in the inactive, GDP bound complex (it gets stuck there)
Translation inhibited because less active eiF2

Question 8

How many eiF2 kinases are there and what are they activated by (examples)

Answer 8

4

stresses: hypoxia, viral infection, DNA damage, aa starvation

Question 9

When is increased eiF2 phophorylation mistakenly found

Give e.g.s

Answer 9

neurodegenerative diseases
Alzheimers, Parkinsons and prion disease
They correlate with high levels of phosphorylated eiF2, as patr of the body’s protective mechanism to suppress translation (neurons are trying to protect themselves from further plaque build up)

Question 10

What is the name of the response where eIF2 is phosphorylated to try and prevent the build up of aggregated protein?

Answer 10

unfolded protein response

Question 11

In mice, how can prion disease be ameliorated?

State the general results of the experiment

Answer 11

Over expression of eif2 Phosphotase

This restored tranlsation, synatptic function increased and so did survival rate

Question 12

What do mutation in eIF2B cause in humans

Answer 12

Vanishing white matter disease
-inherited brain disorder characterised by loss of cerebral white matter
Often precipitated by trauma or stress - consistent with the idea that the pathway is involved in stress response

Question 13

What was the other point of control in initiation? Other than eiF2…?

Answer 13

The cap binding protein, eIF4E

Question 14

Briefly describe the role of eIF4E

Answer 14

Binds to mRNA cap, is on the eIF4G scaffold along with eiF4A and the mRnA in the cap binding complex

Question 15

What happens to the cap binding complex in stress?

Answer 15

It can’t form so translation suppressed

Question 16

How is translation surppressed via the eIF4E protein?

Answer 16

4E-BP binds to it, cap binding complex cannot form

Question 17

what is 4E-BP regulated by itself?

Answer 17

mTOR (a kinase)

When phosphorylated by TOR, 4E-BP is unable to bind to eif4E, so translation can porceed

Question 18

How is mTOR itself controlled

Answer 18

Requires sufficient levels of growth factors, amino acids and energy
This ensures translation is only occuring in favourable conditions, when these are all plentiful (ie. in good condition 4E-BP is phosphorylated)

Question 19

How much of the cell’s energy does lation consume

Answer 19

a whopping 80%

Question 20

What can mTOR be inhibited by and what effect can this have

Answer 20

Rampamycin

Extend life span

Question 21

Outline the experiment that demonstrated the effects of rampamycin on mTOR and the results

Answer 21

MIce given rampa
- male life span increased by 28%, females by 38%
May explain why calorific restriction increases life span - because restricted animals are constantly stressed (and hungry)
- Also protected against age related disorders e.g. cardiovasculardysfunction, neurodegeneration, cancer

But we don;t fully understand the role of mTOR yet

Question 22

Describe ho wmTOR inhibition could be used in cancer therapies

Answer 22

A rampamycin derivative with improved phamokinetcs developed - ‘rapalogues’
-remains in patient longer so better
Clininical trails
-currently approved for some kidney and pancreatic cancers

Question 23

What is a possible reason why mTPR inhibition doesn;t work well for most cancers

Answer 23

mTOR inhibition targets other signalling pathways through feedback loops so upstream kinases may be affected, meaning it has limited effect

Question 24

As well as 4E-BP, what else does mTOR regulate

Answer 24

ribosomal protein S6 and several transcription factors (but protein synth is its major target)

Question 25

What eukaryotic initiation factor is strongly implicated in cancer, when it’s levels have risen

Answer 25

eIf4E

Question 26

What do increased levels of eiF4E in cancer correlate with

Answer 26

decreased survival

Question 27

So does eIF4E definitely have a role in cancer?

Answer 27

NO, could just be a bi-product,

Other components of the protein synthetic machinary are implicated

Question 28

what evidence is there that eIF4E does have a role in cancer

Answer 28

• over expression can induce malignant transformation of human cells in culture, and tumourgenesis in transgeneic mice
• Over expression of $E-BP can counteract this transformation by eIF4E - ie 4E-BP inhibits eIF4E and stops the effects
• raised 4E-BP can mean improved survival prospects

Question 29

What are consistently overexpressed in cancer cells and how do you know

Answer 29

tRNAs and rRNAs (for increased protein synthesis)
-RTPCR - cancer cells give brighter bands than non so more RNA present
Sometimes not elevated in begningn tunours

Question 30

So what is very important for cancer cells

Answer 30

INCREASED PROTEIN SYNTHESIS - accidental caps but is actually that important

Question 31

Why is elevated protein synthesis important for cancer

Answer 31

Without it there would only be cell proliferation not cell growth (increase in mass) as well. In fact mass would get smaller and smaller as more divisions occurred - needs similar accumulation of mass

Question 32

Regardless of the overall rate of protein synthesis, how well are strong mRNAs translated

Answer 32

always well

Question 33

How do weak mRNAs respond to elevated protein synthesis

Answer 33

Respond preferntially
In fact they struggle to get expressed when protein synth is normal
When protein synth levels increase, they receive a disproportionate increase in their own expression

Question 34

Often, what kind of mRNA encodes oncogenes

Answer 34

weak - e.g. c-myc

Question 35

What makes an mRNA ‘weak’

Answer 35

a secondry structure in the 5’UTR which makes in hard for the ribosome to assemble

Question 36

In multiple myeloma (type of cancer - malignancy of plasma cells), what mutations are present?

Answer 36

mutation that stimulates c-Myc (oncogene that encodes TF implicated in lots of cancers) translation

Question 37

What did a small study find regarding the cause of multiple myeloma and why were the results ‘astounding’?

Answer 37

42% of patients had a point mutation in the 5’UTRthat altered a secondary structure there and enhanced ribsomal recruitment to the RNA, therefore increasing lation.
This mutation is NEVER present in a healthy person - very important because normally you would expect SOME sequence variation

Question 38

When do cancers actually surpress lation?

Answer 38

When O2 or nutrients limited, e.g. in the middle of a large tumour

Question 39

How do cancer cells surpress lation? And what type of translation is inhibited, and why is this?

Answer 39

4E-BP expression increased
Only Cap dependant mRNA translation is inhibited
Cap independant translation is not - because many mRNAs translated in this independant manner code for angiogenic factors e.g. Vascular Endothelial Growth Factor. These are immune to 4E-BP
Therefore cancer can slow down general lation but angiogenesis is stimulated - promoting tumour development

Question 40

What does IRES stand for and what does it do

Answer 40

Internal Ribosome Entry Site
The regions of complex secondary structure in 5UTR that allow recruitment of ribosomes independently of cap recognition complex
Determined via folding not sequence

Question 41

HOw does IRES faciliate cap independent lation exactly

Answer 41

Facilitates recruitment of PIC downstream from CAP but upstream from stop codon

Question 42

IN cancer cap dependant lation is repressed, when else is it?

Answer 42

In mitosis - IRES mediated translation contues (but bulk stopped).
The subset of mRNAs that allow mitotic progression are controlled by IRES so still translated

Question 43

Where were IRES first discovered?

Answer 43

In picornovirus (polio) - these degrade eIF4E to surpress the lation of the host, whilst maintaining expression of their IRES controlled IRES, which contain viral RNA.