W11L1 regulation of translation in eukaryotes Flashcards
DNA sequencing and protein sequencing
DNA sequencing technology
- very fast, very cheap, very effective
Protein sequencing
- (relatively) slow, expensive, challenging
DNA sequencing technology can be used to quantify rates of translation
-polysome sequencing
-ribosome-tagging/affinity purification
-ribosome profiling (Ribo-Seq)
Polysome profilling
- extract RNA still bound to ribosome
-run through a sucrose gradient to separate by density
-collect RNA
Trap/ ribotag
- antibody to the ribosome protein
-pulling down the ribosome
-measure RNA
Ribosome profiling
-identify sequence where ribosome bound
- using special nuclease digestion to isolate RNA seq where it is not covered by ribosome
-Tells us position of ribosomes on mRNAs
à which ORFs are being actively translated
Calculating translational efficiency
Translational efficiency= ribosome footprint density/ relative mRNA abundance
Translational regulation under starvation
- many gene translational efficiency and mRNA change does correlate
-but there are some that have higher levels of translation
Method in regulation of translational initiation
Global regulation of mRNAs at 5’cap
Specific regulation of mRNA by 3’UTR
Specific regulation of mRNA by 5’UTR
Regulation of mRNA recognition by TOR kinase
-eIF4F complex required for cap-dependent translation
-growth factor activate mTor kinases which phosphorylates 4Ebp
-p4Ebp cannot bind to eIF4E, translational start
-under low nutrient, TOR kinase become inactivated, 4EBP active, prevent translation
Global vs specific regulation by mTOR
-99% mRNA are translationally regulated by mTOr
-based on ribosome profiling of mTOR inhibitor (torin 1) treated mouse cells with double knock out
-The effect of mTOR inhibition on translation requires 4E-BPs
BUT, some mRNAs are more susceptible to TOR than others
MTOR and IRES
-as IRES doesn’t have a 5’ cap, it is not affected by mTOR
Why is there differences to mTOR susceptability
-due to 5’ TOP sequences affect the binding affinity of 4EBP
mRNA-specific translational repression by a 4E-BP in drosophila
- polar localization of mRNA in drosophila. Oocyte
-Posterior localization of maternal Oskar mRNA establishes polarity in Drosopohila oocyte
-need to repress Oskar during transport
Mechanism of repressing Oskar during transport
-mRNA-specific translational repression by a Cup, a 4E-BP
-Abundance of Cup is too low to repress all mRNAs
-Bruno is required to recruit Cup to Oskar mRNA
- Bruno bind to Bruno respond element in the 3’
Iron homeostasis – balancing deficiency and excess
-Fe is required for function of 100s enzymes, especially oxygenic metabolism
Fe is highly reactive and is toxic to cells
- Ferritin is an iron-storage protein:buffers against toxicity and deficiency
Control of Ferritin
Sufficient iron in cell:
IRP1 = aconitase, an Fe-containing enzyme (Need iron to fuction)
Insufficient iron in cell
Fe-free Aconitase becomes an RNA binding protein
