Feb 27 Flashcards
(21 cards)
What happens to yeast if you give all amino acids except 1?
They will will then make all the amino acids not just that one. If they are given all AA’s, they will not make any
What is Gcn4, what does Gcn stand for?
Gcn is general control nonderepressible. Gcn4 is a transcription factor that is made if yeast is starved of at least 1 AA, this turns on genes for AA biosynthesis
What is Gcn2 and IF2? What does IF stand for?
IF is initiation factor. IF2 is a GTP-binding protein, has GTPase activity, and is phosphorylated by Gcn2 (protein kinase)
What energy source is needed to activate amino acid by attaching it to tRNA? What enzyme does this?
ATP is hydrolyzed to AMP, done by Aminoacyl-tRNA synthetase
What are the steps for initiation of protein synthesis in eukaryotes using eIF2 (eukaryotic initiation factor 2)?
- eIF2B is the GEF for eIF2, so GDP exchanged for GTP=active conformation
2.eIF2-GTP binds to Met-tRNA - Then small ribosomal subunit binds
4.Small subunit binds to mRNA, finds AUG, docks 60S subunit to start elongation
5.GTP hydrolyzed to GDP
How does amino acid starvation cause protein synthesis to stall in yeast?
- If AA starved, the accumulation of uncharged tRNAs will activate Gcn2
- Gcn2 is a protein kinase that PO4s eIF2 on the alpha subunit.
3.When GDP-bound eIF2 binds to eIF2B (GEF), it will lock up, and GTP won’t replace GDP.
4.This stalls initiation, but any protein synthesis that has been initiated will continue
How is Gcn2 responsive to the build up in uncharged tRNA?
It has kinase domain like usual, but has domain that looks like tRNA synthetase that will add AA’s to tRNA, so it has a way of recognizing them, and if accumulate, then kinase domain activated.
What type of phosphatase is Gcn2?
PP1
What is the limiting protein that causes protein synthesis to stall so quickly (eIF2, eIF2B)?
It is the GEF eIF2B, so phosphorylation of only 30% of eIF2 will bind up all the available eIF2B
What sort of stimuli will cause the phosphorylation of eIF2 by not only Gcn2 but other similar protein kinases?
Heat shock, glucose starvation, heavy metals, viral infection, AA starvation etc.
How widespread is Gcn2 and its homologues in eukaryotes?
It is ancient protein, so it has 3 close relatives that will not be activated by AA starvation, but will PO4 the same serine on eIF2.
How does Heme controlled inhibitor (HCI) stall protein synthesis initiation?
Lack of heme is correlated with unfolded proteins in cytosol. HCI responds to unfolded proteins, and will PO4 eIF2 on serine.
How does dsRNA activated protein kinase (PKR) stall protein synthesis initiation? What happens with the active site that gives it this function?
Infection leads to interferon (protein) being produced. Interferon induces PKR transcription. If dsRNA is present, PKR will bind to it and phosphorylate eIF2.
A part of dsRNA binding domain will hang into active site of kinase domain. This AA sequence is pseudosubstrate that looks like the eIF2 substrate, but maybe not serine in the spot so thats why not phosphorylated (psudeosubstrate). Then dsRNA binding to domain moves the pseudosubstrate out of the active site so it can now act on eIF2.
What are a few ways that viruses can counter dsRNA activated protein kinase (PKR) from stalling protein synthesis initiation?
They can keep their own dsRNA bound, they can also dephosphorylate eIF2, or block its phosphorylation somehow.
Give details on HSV-1 gene y134.5 and GADD34?
GADD34 is human protein (growth arrest and DNA damage). It mops up PP1 (protein phosphatase 1) and directs it to dephosphorylated eIF2, so undoing the work of the other 4 kinases that deactivated eIF2. HSV-1 gene 134.5 is a gene in herpes simplex virus that has similar product to GADD34, does the same thing.
What motif do GADD34 and y134.5 share that binds to PP1 and directs it to dePO4 eIF2?
They both have RVXF motif
Following eIF2 subunit alpha phosphorylation, this stalls protein synthesis initiation. How do some mRNAs get preferentially translated to help the cell direct all remaining resources to surviving stress response? What 2 transcription factors?
ATF4 and CHOP are transcription factors that both induce gene to make GADD34, which will recruit PP1 to dephosphorylate eIF2 and let protein synthesis continue.
What is the PP1 variant that maintains basal eIF2 dephosphorylation and lets protein synthesis initiation to continue when stress not present?
It is CReP, it has C-terminus like GADD34, so it directs some PP1 that is part of it to keep eIF2 alpha subunit dePO4’d
What does GADD34 have for domains on it that will help it do its function well?
It has RVxF motif so binds to PP1, then it has domain so it is expressed in ER membrane.
How does PERK stall protein synthesis activation? What is special about it?
PERK is present in the ER MB, it is fusion protein. It detects unfolded proteins in ER lumen( indicating ER stress) with Ire1 domain, then has Gcn2 part that will phosphorylate eIF2 on the cytosolic side of ER.
