MARIA - LECTURE 4 Flashcards
how does eIF2 discriminate between the initiator tRNA met and the normal tRNA met?
eIF2-GTP has high affinity for that initiator tRNA, binds it in the right conformation and stabilizes it
the normal tRNAmet has a different nucleotide sequence in the stem, which makes the binding different and therefore does not recognise it
how does prokaryotic elongation happen
EF-Tu brings tRNA into the A site, hydrolyses GTP and leaves
peptide bond is formed
EF-Ts recycles EF-Tu GTP, needed as EF-Tu affinity to GDP is high
EF-G binds at the same spot as EF-Tu (gives an order) and hydrolyses a GTP to cause translocation of the ribosome
EF-G-GDP does not need recycling with the help of another factor because the affinity of EF-G to GDP is low and is high for GTP
energetic cost of elongation: 2 GTPs/amino acid after methionine
how does eukaryotic elongation happen
- eF1alpha brings tRNA into the A site, hydrolyzes GTP and leaves
- eEF-1betagamma replaces the GDP on eEF-1alpha with GTP:
first eEF-1betgamma itself binds to replace the GDP on eEF-1alpha
then, GTP arrives and eEF-1betgamma is released - peptide bond is formed
- eEF-2 hydrolyses GTP to cause translocation of the ribosome
eEF-2 binds the same site as eEF-1alpha
2 GTPs are hydrolyzer per AA added, but none of this energy is directly used to form a peptide bond
what is the process called in which errors are avoided during elongation?
accomodation
error rate during elongation: 10^3-10^5
ribosomes check the correct pairing between codon and anticodon on the A site
can be correct by perfect watson-crick pairing or wobble pairing
how does accommodation work
what are antibiotics that target translation and how they work
how does the combination of chloramphenicol to a macrolide work
chloramphenicol binds to the A site and blocks the peptidyl transferase center
macrolides block the entrance to the exit channel, interferes with the elongating polypeptide
how does ricin work
it is a ribosome inactivating protein (RIP)
has an N-glycosylase activity and depurinates nucleotides
abolishes the interaction between the 60S ribosome and eEF-2, so ribosomes cannot translocate
how does the diphteria toxin work
it is another RIP
diphteria toxin (DT) has a T, C and R domain
enters the cell, and in the endosome the pH drops
this causes the cleavage of the C domain, which will go carry out the toxic function of DT
histidine699 on eEF2 is modified into diphthamide (important for ribosome binding)
diphthamide will be modified by DT to have an ADP-ribosyl group attached to it, which will lead to cell death
what is an example of RIPs used as anticancer agents
ontak: IL-2 and diphtheria toxin fusion protein
a subset of leukemia and lymphoma cells express hugh affinity for IL-2 receptors
this will target these cells and induce their death with DT
how does prokaryotic termination work
how does eukaryotic termination work
what is the structure of eRF-1 and what does it look like
how does the peptide-tRNA hydrolysis happen
done by eRF-1 on the GGQ motif end
how does eRF-1 recognise the stop codon
this happen on the NIKS domain (anticodon like domain)
first binding pocket it only small enough for pyrimidines: the only options are U and C
U is better than C because it can engage in hydrogen bonding
the options for the second binding pocket are G or A: both purines are stabilized by hydrogen bonding stacking and Glutamine 55
therefore if it stabilizes with 2 As (UAA) that works, or one A (UGA/UAG) that also works
if there isn’t an adenine (UGG or anything else) it will not be recognised)
what do aminoglycosides do
interfere with accomodation
in a drug screen using firefly luciferase as a reporter gene, which drug was found to be able to help the ribosome make the full protein despite the presence of a premature stop codon
PTC124
how does Duchenne Muscular Dystrophy work?
DMD: X linked recessive
mutation in dystrophin
premature stop codon leads to the formation of a shortened dystrophin protein
dystrophin is responsible for giving shape to muscle cells, anchoring to actin filaments and providing extracellular communication
no dystrophin means muscle cells get full of water, explode, leading to cell death
what is the structure of actively transcribed mRNAs
mRNAs circularize
so that ribosomes finish and find the cap directly after, if it is in close proximity to the cap
eIF-4G is a scaffold that helps fold it together
eIF4G has a domain that binds poly A binding protein
however, this model has been challenged
how do histone mRNAs circularize despite not having a poly A tail?
histone mRNAs are only made during DNA synthesis: G1, S, G2
the only time that new histone proteins will be needed
histone mRNA doesn’t have a poly A tail but there is a stem loop structure, followed by ACCCA
stem loop BP binds this stem loop, out of nucleus it goes
it is then translated, since SLIP, which binds SLBP, interacts with eIF4G
at the end of S phase, SLBP is degraded
