Lecture 24 - Translation Continued Flashcards
What was the VERY DRAMATIC EXPERIMENT that bob refered to ie what was the dramatic conclusion
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
Describe the very dramatic experiment
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
What are the 2 key points of control in euk translation initiation
assembly of cap binding complex
eIF2 - ie the deliverance of tRNA to the PIC
What eIF2 does (very brief because it all in previous lecture)
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
How is eIF2 recycled?
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
What condition prevent the recycling on eIF2 and therefore surpress ttranslation?
Stress.
What impact does stress have on eIF2
eIF2 phosphorylated by eIF2 kinases, sequestering eiF2B in the inactive, GDP bound complex (it gets stuck there)
Translation inhibited because less active eiF2
How many eiF2 kinases are there and what are they activated by (examples)
4
stresses: hypoxia, viral infection, DNA damage, aa starvation
When is increased eiF2 phophorylation mistakenly found
Give e.g.s
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)
What is the name of the response where eIF2 is phosphorylated to try and prevent the build up of aggregated protein?
unfolded protein response
In mice, how can prion disease be ameliorated?
State the general results of the experiment
Over expression of eif2 Phosphotase
This restored tranlsation, synatptic function increased and so did survival rate
What do mutation in eIF2B cause in humans
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
What was the other point of control in initiation? Other than eiF2…?
The cap binding protein, eIF4E
Briefly describe the role of eIF4E
Binds to mRNA cap, is on the eIF4G scaffold along with eiF4A and the mRnA in the cap binding complex
What happens to the cap binding complex in stress?
It can’t form so translation suppressed
How is translation surppressed via the eIF4E protein?
4E-BP binds to it, cap binding complex cannot form
what is 4E-BP regulated by itself?
mTOR (a kinase)
When phosphorylated by TOR, 4E-BP is unable to bind to eif4E, so translation can porceed
How is mTOR itself controlled
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)
How much of the cell’s energy does lation consume
a whopping 80%
What can mTOR be inhibited by and what effect can this have
Rampamycin
Extend life span
Outline the experiment that demonstrated the effects of rampamycin on mTOR and the results
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
Describe ho wmTOR inhibition could be used in cancer therapies
A rampamycin derivative with improved phamokinetcs developed - ‘rapalogues’
-remains in patient longer so better
Clininical trails
-currently approved for some kidney and pancreatic cancers
What is a possible reason why mTPR inhibition doesn;t work well for most cancers
mTOR inhibition targets other signalling pathways through feedback loops so upstream kinases may be affected, meaning it has limited effect
As well as 4E-BP, what else does mTOR regulate
ribosomal protein S6 and several transcription factors (but protein synth is its major target)
What eukaryotic initiation factor is strongly implicated in cancer, when it’s levels have risen
eIf4E
What do increased levels of eiF4E in cancer correlate with
decreased survival
So does eIF4E definitely have a role in cancer?
NO, could just be a bi-product,
Other components of the protein synthetic machinary are implicated
what evidence is there that eIF4E does have a role in cancer
- 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
What are consistently overexpressed in cancer cells and how do you know
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
So what is very important for cancer cells
INCREASED PROTEIN SYNTHESIS - accidental caps but is actually that important
Why is elevated protein synthesis important for cancer
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
Regardless of the overall rate of protein synthesis, how well are strong mRNAs translated
always well
How do weak mRNAs respond to elevated protein synthesis
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
Often, what kind of mRNA encodes oncogenes
weak - e.g. c-myc
What makes an mRNA ‘weak’
a secondry structure in the 5’UTR which makes in hard for the ribosome to assemble
In multiple myeloma (type of cancer - malignancy of plasma cells), what mutations are present?
mutation that stimulates c-Myc (oncogene that encodes TF implicated in lots of cancers) translation
What did a small study find regarding the cause of multiple myeloma and why were the results ‘astounding’?
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
When do cancers actually surpress lation?
When O2 or nutrients limited, e.g. in the middle of a large tumour
How do cancer cells surpress lation? And what type of translation is inhibited, and why is this?
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
What does IRES stand for and what does it do
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
HOw does IRES faciliate cap independent lation exactly
Facilitates recruitment of PIC downstream from CAP but upstream from stop codon
IN cancer cap dependant lation is repressed, when else is it?
In mitosis - IRES mediated translation contues (but bulk stopped).
The subset of mRNAs that allow mitotic progression are controlled by IRES so still translated
Where were IRES first discovered?
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.
