Translation Flashcards
How many different codons and the start and stop codons?
64 different codons. AUG for start. UAA, UAG, UGA (also selenocysteine the 21st amino acid.
Describe the features of a typical mRNA chain.
A 7-methyl-GMP cap with a 5’ to 5’ linkage. 5’ UTR (which contains IRE for certain proteins), a coding region, then 3’ UTR which is longer and contains a lot of info regarding RNA structure. THe poly A -tail is a regulatory site and is non templated.
Describe the tRNA and aminoacylation.
It has a 3’ amino acyl end where the amino acid is attached to with an anticodon which binds to the mRNA. The 3’ end is always a CCA with A at the 3’ end. Amino acyl tRNA synthetases require ATP to add on an amino acid. These proteins have a synthesis site and an editing site so that if an incorrect amino acid is added to the tRNA, the editing site binds and removes the tRNA. Each tRNA synthetase only recognizes a particular amino acid. Histidinyl tRNA synthetase would only recognize histidine.
The ribosome (60S and 40S) is a ribozyme. Describe what else the ribosome has.
Peptide bond formation doesn’t require the interaction of any protein. It has 3 binding sites for tRNA. E, P, A in that order.
Describe initiation.
The small ribosomal subunit has the initiator tRNA bound with elF2 (which delivers the initiator) which is bound with GTP. More factors arrive. elF4E binds to the 5’ cap of the mRNA. Then elF4G binds to elF4E and serves as a scaffolding protein and bends the transcript. This will form the pre-initiation complex when it comes in contact with the mRNA. ATP hydrolysis will start the scanning of the mRNA until it reaches the first AUG. The large subunit arrives placing the initiator tRNA in the P site. This is the switch for GTP hydrolysis and the dissociation of the eIF2 and other factors.
Describe elongation.
eEF1A, which binds to every subsequent tRNA brings the tRNA into the A site, and is itself bound to GTP. GTP hydrolysis allows the two to be close enough together so the two can peptide bond. Then eEF2 comes in and assists with translocation, pushing the tRNA from the A site into the P site. This also carries GTP so the hydrolysis dissociates eEF2 from the complex. During this the A site will proofread.
Describe termination
There is no terminator tRNA, the stop codon binds with eRF1 which binds and cleaves the peptide chain off releasing the peptide.
What are differences between prokaryotic and eukaryotic translation.
Prokaryotes:
their mRNA is polycistronic (mRNA encodes many genes) their mRNA has no modifications, The Shine Dalgarno sequence is what orients the ribosome to initiate translation, we can use selective inhibition because their ribosomes are sufficiently different (70 S ribosome, 30 and 50S.
Post transcriptional but pre-translational regulation: RNA editing.
Apolipoprotein B - apoB mRNA is edited to introduce a stop codon so a shorter protein is made for use in the small intestines vs the liver.
Describe miRNAs
They are small RNA molecules that bind to mRNA and regulate translation. They usually bind to the 3’ UTR forming RISC complexes which physically impede translation initiation (because of the bend). THe ribosome recognizes both 3’ and 5’ ends simultaneously.
Iron homeostasis.
5’ UTR for example the ferritin mRNA which stores iron has an IRE which can bind IRP. IRP will only bind if there is low iron (no iron to store). This physically impedes translation, inhibiting translation of ferritin proteins.
How does nutritional status regulate translation.
Essentially it is all about elF2 being phosphorylated which can be due to low amino acid concentration in the case of mall nutrition but also for many other stresses.
So elF2 after initiation is still bound to the GDP it has a high affinity for. In order to be recycled there needs to be a Guanine Nucleotide Exchange Factor (GEF) which exchanges GTP with the GDP. That is what eIF2B does. However in cases of stress, the eI2F is phosphorylated. This will bind to elF2B and sequester the proteins because el2F is in more abundance. Lack of elF2B leaves el2F in its inactive form and protein syntehsis stops dramatically.
How does hypoxia regulate translation?
Hypoxia regulates translation through the mTOR pathway which is the pathway that oversees growth. The protein of interest is the elF4E protein which binds 4G and binds 5’ cap. The protein of interest is the 4EBP. It will only bind if it is dephosphorylated and then inhibit translation. So in hypoxia, 4EBP (which is usually half phosphorylated is now completely dephosphorylated, binds to elF4E and inhibits translation.
So wtf does selenocysteine do?
It is used to make deiodinases which are subunits of the thryoid hormones. mutations with selenocysteine interferes with the production of thyroid hormone and thus lead to hypothyroidism. The SBP2 protein is what makes the UGA read as a selenocysteine rather than a stop codon. So mutations in that have this effect.
What does dipthera toxin do.
The A fragment enters the cell and inhibits eEF2, the translocation protein. Translation stops, you will die.
