CBG Lecture 7: Translation Flashcards
what is the ribosome
a massive ribonuceloprotein with ribozymal peptidyl transferase activity
what Svedberg unit are bacterial ribosomes
70S
what Svedberg unit are eukaryotic ribosomes
80S
where does translation occur
on the ribosomes
what does translation require
ribosome
charged tRNA, mRNA GTP and variety of initiation, elongation and termination factors to ensure accurate synthesis
what do ribosomes do
translate the codons of the mRNA into the sequence on a protein
how many subunits in a ribosome? what are they called
2 subunits
Large subunit
Small subunit
what does SSU do
small ribosomal subunit
binds mRNA, ensures codon pairs correctly with anticodon
what does LSU do
large ribosomal subunit
AS peptidyl transferase found here
where aas are attached to make a protein
what is the catalytic site of a ribosome made of?called?
peptidyl transferase active site
made entirely of rRNA
its a ribozyme
what is the error rate for translation of eukaryotes/bacteria
10-4 per aa
which translates faster, E.coli or humans?
E.coli by 10 times
E.coli : 20aa per sec
in prokaryotes and euks where does translation occur
in cytoplasm
WHAt machine is used for translation
ribosome, ribozyme, LSU, SSU
how is translation initiated
Initiation factors recruit tRNAi to the P site on the naked SSU, expending GTP
how many sites are on the SSU?
3
E(xit site)
P(eptidyl tRNA site)
A(minoacyl tRNA site)
what does the E stand for on the SSU
exit site
what does P stand for on SSU
peptidyl tRNA site
what does the A stand for on SSU
aminoacyl tRNA site
what is initiatior tRNA charged with in bacteria and endosymbionts
formylmethionine
what is initiator tRNA charged with in eukaryotes and archaea
methionine
whereabouts is the adenosine in tRNA
at 3’ end
how is initiator tRNAi removed? when is it removed
removed by peptidase after translation
outline initiation for translational initiation (inc. elF etc)
elF3 prevents LSU binding
elF1 blocks A site
elF2 brings tRNAi to P site
expends GTP, hydrolysis GTP, releases IFs allows LSU to bind
outline elFs and what they do in translational initiation
elF3- prevents LSU binding
elF1 - blocks A site
elF2 - brings tRNAi to P site, , hydrolysis GTP, releases IFs allows LSU to bind
what part of mRNA does the ribosome bind to first in eukaryotes
the methylguanosine 5’ cap, then finds the first AUG after that
the eukaryotic consensus (Kozac sequence) plays similar role to Shine-Dalgarno sequence but not as conserved
what does elF3 do
prevents LSU binding
what does elF1 do
blocks A(minoacyl tRNA) site
what does elF2 do
brings tRNAi to P(eptidyl tRNA) site, hydrolysis GTP, releases IFs allows LSU to bind
what is a Kozac sequence? what is it analogous to
eukaryotic consensus
similar role to SDS (Shine Dalgarno Seqeunce) but not as strongly conserved
how is the AUG in prokaryotes distinguished from other AUGs
by a Shine Dalgarno Sequence
why is AUG ambiguous in bacterial mRNA
because their mRNA is polycistronic, so AUG could be a start codon, inernal methionine or out of phase codon with reading frames
why do eukaryotic mRNAs not need an SDS
because its monocistronic, ribosome just needs to bind mRNA 5’ cap then find the AUG after
what is the chance of getting an SDS sequence in polycistronic mRNA
1/262144
what is the chance of getting an AUG codon
1//64
what provides evidence for LUCA
genetic code is almost universal
AUG is the typical start codon
there are only a small number of variations of genetic code
how many GTPs are expended per aa in translational elongation
what is it used for
2GTP/aa - energy intensive
transfers the peptidyl on the P site tRNApeptidyl to the A site tRNAaminoacyl
outline elongation factors and their roles used in translational elongation
EF1 brings charged tRNA to A site
EF2 shunts ribosome along
LSU catalyses peptide bond formation
what does AUG code for in bacteria? in eukaryotes?
AUG - methionine in euks
formylmethionine in proks
what can UGA also code for
selenocysteine instead of a stop
how does ribosome distinguish between whether something is a stop codon or codes for an aa
based on secondary structures in mRNA
how is the initiator aa removed from the mature protein
by a peptidase
what does tRNA wobble lead to
non-Watson-Crick pairing at 3’ third base of codon
how many aas? how many tRNAs
20 aas
40 tRNAs
what are stop codons bound by
release factors (tRNA peptidyl hydrolase) terminating translation
what does EF1 do
brings charged tRNA (eg. serine or threonine) to A site
name some charged tRNAs
serine
threonine
(have OH group)
what does EF2 do
shunts ribosome along - conformational change
uses GTP
what part of ribosome catalyses peptide bond formation
LSU
what does the LSU do
catalyses peptide bond formation
what part of translational elongation requires ATP
2 ATP to attach aminoacyl to the tRNA in first place
for every amino acid added = 4 ATP
how many GTPs and ATPs used during translational elongation
2GTP per AA
4ATP per AA
how can mRNA convey meaning
through secondary structures
eg. convert stop to selenocysteine (UGA)
convert stop to pyrolysine (UAG)
what does UGA
UAG code for
UGA - stop/selenocysteine
UAG - stop/pyrolysine
how does mRNA convey meaning to ribosome about UGA/selenocysteine
converts STOP to selenocysteine by expending GTP
catalysed by a SEcIS : selenocysteine inserion element
what is a polyribosome array
what does it do
lots of ribosomes
acts on a elF4-coiled mRNA to increase efficiency of translation
how is protein folding detemined
by primary structure
what is a molten globule
native secondary structure by liquid interior
what is native state
the favoured entropic state but can get trapped in local energy minima
give some examples of disease caused by faulty protein folding
CF
Marfan syndrome
Creutzfeld Jakob disease
Alzheimer’s disease
how is CF caused
misfolding of transmembrane regulator
how is Margan syndrome caused
misfolding of fibrillin
how is Creutzfeldt Jakob disease caused
misfolding of prion protein due to extracellular aggregation
how is Alzheimer’s disease caused
misfolding beta-amyloid due to extracellular aggregation
what does a stacked beta sheet in a misfolding prion to
catalyses the misfolding of other prions leading to an exponential fatal misfolding catastrophe
what are chaperonins
protein chaperones help fold proteins
where does protein folding take place - is it an active process? how does it work
inside barrel of GroEL/GroES chaperonin - uses ATP
exposed hydrophobic residues on outside of protein are relocated to inside of protein
what happens to unfoldable and unwanted proteins
theyre ubiquitinated and degraded in proteasomes
what are proteasomes
they ubiquitinate and degrade unfoldable and unwanted proteins
how do proteasomes ubiquitinate
attach a 5 ubiquitin tail to the waste protein
how many subunits are present in proteasome
2
regulatory subunit: recognises 5Ubiquitinated protein, deubiquitinates and unfolds it
proteolytic subunit: degrades the protein
what does the regulatory subunit of a proteasome do
recognises 5 U ubiquitinated protein, de-Us it then unfolds it
what does the proteolytic subunit of a proteasome do
degrades the unwanted protein
