Translation Flashcards
Define translation?
Process by which the genetic message, encoded by RNA bases, is expressed in the form of an amino acid sequence in a protein using the 20 amino acids
What is required for protein synthesis
template - mRNA building blocks - amino acids a way to assemble a chain - ribosomes rules on decoding - aminoacyl tRNAs energy - ATP x 4 for aa
Outline ribosome structure?
Small and large subunits need to join together to form 3 sites for tRNA to occupy.
mRNA slides through a channel which is on small subunit.
Composed of rRNA and proteins.
Ribozyme.
Outline translation initiation?
Initation factors - pro IFs euk eIFs
Positioning of the small ribosomal subunit and first aminoacyl tRNA at the initiation codon.
Join large subunit
Slowest step - limits rate of translation
Outline translation elongation?
Brings the next tRNA, joins together. Expels first tRNA, cycle repeats.
How fast is translation?
10-40 aa s-1
error rate of translation?
1 in 10,000
Outline translation termination?
Release factors - pro RFs euk eRFs
Release the completed polypeptide when stop codon is reached - no tRNA corresponding to the stop codon
Brings in a factor to release the completed polypeptide
What factors release the completed polypeptide?
pro RRF
euk ABCE1
prokaryote ribosomal subunits?
30S, 50S = 70S
How is the prokaryotic initiation complex formed?
30S subunit binds to mRNA (binds a special formylmethionine tRNA) at the P site using initiation factors IF1, IF2, IF3 and GTP
IF1?
binds in A site, prevents elongator tRNAs from entering
IF2?
binds GTP and fMet-tRNA
IF3?
prevents association with the 50S, ensuring fidelity of the initiation codon selection
what is the Shine Dalgarno sequence?
on prokayotic mRNA.
base pairs to the 3’ end of 16S rRNA. Places the start codon (AUG) at the P site, about 10 bases 3’ of the S-D sequence
what does the Shine Dalgarno sequence allow?
the ribsomal subunit can stay attached and look for next sequence in polycistronic mRNAs
what follows the binding of Shine Dalgarno sequence?
followed by binding of 50S subunit and dissociation of IF1 and IF3. binding causes hydrolysis of the GTP on IF2, causing it to also dissociate.
differences in eukaryotic mRNA for initation?
a cap and poly(A) tail
no S-D sequence
5’ and 3’ UTRs contain several sequences that help regulate protein expression/mRNA stability & localisation
mRNA is circularised
complexity adds several opportunities for control
what is eIF3?
binds to the cap effectively
binds eIF4G (which is cap bound) and 40S subunit
13 subunits
what is eIF4G?
acts as a link between poly(A) binding protein (PABP) and the cap binding protein
stages of eukaryotic initiation?
eIF2 + 40S + met-tRNA + GTP bind
40S binds to mRNA with other eIFs which are bound to cap and poly(A) tail
40S scans the mRNA looking for AUG codon
what makes codon searching more efficient?
If its within Kozak Consensus
what happens after codon is found?
eIFs dissociate and 60S subunit binds
initiation similarities?
eIF2 is bound to GTP and brings in first tRNA
eIF3 and eIF1 ensure the accuracy of initiation and prevent association of large subunit
when is most protein produced?
when both a cap and tail are on the mRNA
more than cap + tail so must be synergism
makes it circularised so easier for ribosome to find the mRNA again
control of eIF4E availability?
4E-4G interaction is required to allow the ribosome to attach to mRNA
4E-BP1 has a similar sequence motif to the eIF4G.
If lots of growth and nutrition factors –> mTORC1 (kinase) is active so can phosphorylate 4E-BP1 so can’t bind to 4E
control of eIF2 availability?
Brings in tRNA. Key control point.
When translation is initiated, GTP –> GDP + Pi. eIF2B phosphorylates the GDP back to GTP.
When there are stresses on the cell, eIF2B gets blocked. Kinase gets activated and phosphorylates the alpha. stops translation
activation of PKR?
Virus infected cells release interferon. Induced PKR expression in neighbouring cells.
PKR = Protein Kinase RNA dependent
When PKR is expressed, dsRNA is expressed. PKR dimer autophosphorylates and then phosphorylates eIF2a. Reduces protein synthesis, reducing further infection
protein folding?
some proteins require assistance from chaperone. Prevent illicit liasions between proteins.
Misfolded are rapidly degraded.
PERK sits on ER and connects to cytoplasm. Can block protein processing.
Normally BiP binds to PERK, keeping it in an inactive monomeric state. BiP dissociates and binds to unfolded proteins which activates PERK dimers. Inhibits translation until the folding issue is sorted.
Wolcott Rallison disease?
loss of PERK function
codon usage bias?
preference to decode certain codons
how could you make bacteria more effective at producing human proteins?
tweak the codons in the transformed DNA so the codons are what the bacteria prefers
could introduce human tRNAs into the bacteria
describe the adapter molecule?
tRNA
70-90 bases
L shape
3’ terminus ends in CCA with each amino acid linked as an ester to 3’OH
anticodon that recognises the codon through antiparallel base pairing
wobble?
ability of the first base of an anticodon to base pair with more than one codon.
allows an aminoacyl-tRNA to dock with more than one codon
what can U bind with?
G
what can inosine bind with?
A, U or C
looks like guanosine without an amino group but is made by deamination of adenine
which direction are proteins synthesised in?
amino –> carboxyl
features of 30S subunit?
16S rRNA and proteins S1-19
Contains decoding centre.
Helix 44 of rRNA forms A and P tRNA binding sites
3’ of rRNA complements the Shine Dalgarno sequence
features of 50S subunit?
23S rRNA (forms 6 domains) and 5S rRNA and proteins L1-31
Contains peptidyl transferase centre and exit tunnel
A site?
aminoacyl-tRNA
P site?
peptidyl-tRNA
E site?
deacylated tRNA
how is peptide bond created?
NH of incoming amino acid (tRNA) to attack CO on the growing polypeptide which is also still attached to tRNA
have to break the ester bond between the amino acid and tRNA. Nucleophilic attack by the amino group of incoming aminoacylated tRNA
tRNAs do the attacking and formation
how do ribosomes work?
decrease the activation energy needed for peptide bond formation, don’t work by chemical catalysis
EFTu?
Aids aminoacyl tRNA binding. If correct then GTP is hydrolysed
EFTs?
GEF that recycles EFTu.GDP to EFTu.GTP
EFG?
Drives translocation step, requires GTP hydrolysis
Ribosome exit tunnel?
Protects new chain from inappropriate interactions, can sample multiple conformations
