Translational Control Flashcards
Where are proteins synthesised in eukaryotes?
polyribosomes
What does each ribosome subunit do?
- large (60S) - catalyses peptide bond formation
- small (40S) - decodes the genetic message
What are components of a polysome?
- mRNA template
- ribosomes
- tRNAs
- various protein factors
- nascent polypeptides
Describe the ribosome scanning model
small ribosome subunit scans for first AUG codon in favourable context (Kozak consensus)
Where does translational initiation occur from?
the first AUG codon
What are the 5 steps of translational initiation?
1.tRNA binds to elF2 and small ribosome subunit (P site)
2. binds to the mRNA cap preoccupied with initiation factors elF4E and elF4G
3. scans for the first AUG
4. elF2 dissociates and large ribosome subunit binds
5. second aminoacyl-tRNA is added with a correct anticodon
What does each ribosome have?
3 binding sites for tRNAs and 1 binding site for mRNA
What 4 components does a tRNA have?
- acceptor stem - amino acid attachment site
- D arm/loop - recognition site for aminoacyl-tRNA synthetase
- T arm/loop – recognition site for ribosome
- anticodon – pairing with codon
What is a codon?
a sequence of 3 consecutive nucleotides in a DNA or RNA molecule that codes for a specific amino acid
What do codon wobbles positions allow?
one tRNA to recognise multiple codons
What are the 5 steps of elongation?
- aminoacyl-tRNA molecule binds to a vacant site A on the ribosome
- peptide bond formation
- large subunit translocates, leaving the 2 tRNAs in hybrid sites
- small subunit translocates, carrying its mRNA 3 nucleotides through the ribosome
- this resets the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA to bind
How is each amino acid added to the growing polypeptide chain selected?
by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain
What direction is mRNA translated?
5’ → 3’ with the N-terminal end of a protein being made first
What do translation factors do?
drive translation in the forward direction
What does Ef-Tu do?
provide opportunities for proofreading of the codon–anticodon match which allows incorrectly paired 5′ tRNAs to be selectively rejected, and the accuracy of translation is improved
What does binding of EF-G to the ribosome and GTP hydrolysis cause?
a conformational change of the ribosome which moves tRNA to the P-site after formation of the peptide bond
What are the 3 steps of proofreading by 16s rRNA?
- 16S rRNA recognises correctly paired anticodon-codon
- triggers GTP hydrolysis by EF-Tu
- EF-Tu is released to proceed for protein synthesis
How is translation terminated?
by the binding of release factor to an A site bearing a stop codon
What are the 3 eukaryotic termination codons?
UAA, UAG and UGA
What are the 4 steps in the creation of functional proteins?
- protein folding
- binding of small molecule cofactors for proper folding
- covalent modifications
- formation of complex
How are proteins folded into a stable 3D structure?
by burying most hydrophobic residues into an interior core through a large number of non-covalent interactions between various parts of the protein
What happens to incorrectly folded polypeptides?
they are destroyed by proteasomes
What do many newly released proteins have?
molten globules which can be further folded
What does the protein folding pathway use?
the molecular chaperones Hsp70 and Hsp60
What do Hsp70 and Hsp60 do?
- work with a small set of associated proteins
- bind to exposed hydrophobic patches on incompletely folded proteins
- hydrolyse ATP
When do Hsp70 and Hsp60 act respectively?
- Hsp70 - before translation is completed
- Hsp60 - after completion of protein synthesis
What are the 4 steps of the Hsp70 protein folding pathway?
- Hsp70 binds to a string of 4-5 hydrophobic amino acids
- it hydrolyses ATP to ADP and clamps down tightly on target
- rebinds ATP and releases target protein
- incorrectly folded protein may be tried again by the Hsp70 machinery
What are the 7 steps of the Hsp60 protein folding pathway?
- Hsp60 forms large barrel-shaped structure
- captures a misfolded protein by hydrophobic interaction with the exposed surface
- ATP binding
- addition of a cap protein (GroES) to increase the dimension of the barrel rim
- incubation for ~10s
- ATP hydrolysis to weaken cap binding
- additional ATP binding to eject folded protein
What is the proteasome made up of?
- 20S core – multiple subunits that form a cylindrical stack of 4 heptameric rings and active sites on the interior surface
- 2 19S caps – complex of ~20 subunits that recognises ubiquitinated and unfolded proteins
Where is the proteasome present?
throughout the cytosol and nucleus to attack ubiquitin labelled proteins
What is the proteasome?
a compartmentalised protease with sequestered active sites
What are the 3 steps of proteasome action?
- improperly folded proteins are targeted by attachment of a stretch of ubiquitin
- ubiquitin-tagged proteins are translocated to proteosomes, and ubiquitin is removed from the cap by ubiquitin hydrolase for recycling
- targeted proteins are unfolded in the ring at the cap and threaded into the core (cylinder) for degradation
What is the difference between a proteasome and a common protease?
protease cuts once for each binding and does not require ATP whereas the proteasome cuts the entire protein substrate multiple times into short peptides
What do many unstable mRNAs have?
AU rich sequence in their 3’ UTRs
What prevents translation from taking place?
deadenylase shortens the polyA tail
What do actively translated mRNAs tend to have?
longer half-lives
What does codon composition affect?
polyA tail length, translation efficiency and mRNA stability
What do mRNAs enriched in optimal codons have compared to genes enriched in non-optimal codons?
longer polyA tails, higher translation efficiency and higher stability
What are the 2 codes interpreted by the ribosome within the mRNA?
- genetic code which specifies the amino acid sequence
- conserved codon optimality code that shapes mRNA stability and translation
Give examples of specialised mRNA decay mechanisms
- iron starvation
- excess iron
What are miRNAs?
small RNAs transcribed by RNAPII with a cap and polyA tail
What do miRNAs do?
target specific mRNA by base pairing which leads to degradation of mRNAs or translation block
What are the 4 steps of mRNA degradation by miRNAs?
- miRNAs transcribed by RNAPII and exported to cytoplasm
- dicer (RNase) cleaves miRNA to retain the targeting miRNA
- targeting miRNA forms RISC with Argonaute and other proteins
- RISC targets specific mRNAs based on base-pairing
