MARIA - LECTURE 5 Flashcards
Translation in the Mitochondria, Mechanisms to Study Translation
characteristics of mitochondria genome
one circular genome
2/10 copies of it per mitochondria
what does the mitochondrial genome encode?
most of the machinery needed for translation, and not much else
22 tRNAs, 2 rRNAs (12S and 16S) and 13 mRNAs encoding OXPHOS subunits
most mt proteins originate from the nucleus
all mRNAs encoded by mtDNA are translated in the mitochondria
characteristics of mitochondrial mRNA
no 5’ cap
mRNAs are polyadenylated
also no shine dalgarno sequence
characteristics of mitochondrial mRNA translation
there is no glutamine amino acyl tRNA synthetase
how does mitochondrial mRNA translation happen (step by step)
- the two subunits have an affinity for each other, but mtIF3 binds to the small subunit to keep it separate from the big one
- mtIF2 bound to GTP brings the initiator formyl methionine to the P site
- mtIF3 and mtIF2 bound to GDP are released, and the large subunit comes and binds
- mtEFTu bound to GTP binds to tRNA and brings to the A site of the ribosome, another hydrolysis of GDP
- mtEFTs reverts the GTP to GDP
- mtEFG1 bound to GTP translocates the ribosome so that it advances (another GTP consumption)
- tRNA on the exit site now leaves
- mtRF1a (release factor) bound to GTP goes to A site and cuts the bond between the polypeptide chain and the tRNA
- mtEFG2+GTP and mtRRF weaken the interaction between the two subunits
what is the nefarious effect of some antibiotics
some antibiotics that target prokaryotic translation also inhibit mitochondrial translation
what is the rate limiting step of translation
translation initiation is the limiting factor
that is because eIF4E concentration is very low
what are the three ways to measure translation efficiency
- polysome profiling
- ribosome footprinting
- metabolic labelling
what is the average weight of one mRNA, one ribosome and a polysome (20 ribosomes)
how does polysome profiling by fractionation work and the problems with this technique
density gradient fractionation (centrifugation) separates based on weight
heavier polysomes will go down more
measure the optical density
how does ribosome footprinting work
the footprints are then sent for deep sequencing
how does metabolic labelling work
add 35 S-methionine/cystein for a short time period (15mins)
labels only the proteins currently being synthesized
prepare the extracts to quantize radioactivity and analyse on an SDS-polyacrylamide gel
after the adding the radioactivity, boil in 10% TCA, which will wash off 35S-methionine, precipitate the protein onto the filter and hydrolyse aa-tRNA ester linkage
wash and dry and count radioactivity
how do translation rates change during poliovirus infection
how does the profile of synthesized proteins change during poliovirus infection
which protein is cleaved during poliovirus infection
what is the bicistronic assay for identifying internal ribosome entry sites (IRES)
what is type I/II IRES mediated translation
what is type III IRES mediated translation
what is type IV IRES mediated translation
how does type IV IRES - CrPV work
