Translation Flashcards
In what type of particle does eukaryotic mRNA exist in? Why is this important?
It exists as a ribonucleoprotein particle, which allows it to attach to cellular structures so that it can be specifically localised in the cell. The RNA binding proteins in this particle protect the mRNA against degradation.
What is the structure of tRNAs?
Has an anticodon loop responsible for specificity, and at the 3’ end has an amino acid attachment site (CCA sequence). This area where the AA is loaded is composed of an acceptor stem, discriminator base, and the 3’ acceptor end.
What sequences is the start AUG methionine codon within in prokaryotes and eukaryotes?
Prokaryotes- Shine-Dalgarno sequence
Eukaryotes- Kozak sequence
How do amino-acyl tRNA synthetases load an amino acid onto tRNAs?
The synthatase first binds ATP and the specific amino acid, and adenylates by the hydrolysis of ATP and replaces the OH on the amino acid with an AMP molecule. This creates an aminoacyl-adenylate, which can then bind to the appropriate tRNA molecule’s D arm on the 3’ end of the molecule.
What are ribosomes made of?
Proteins that have no enzymatic activity make up the skeleton of the ribosomes, and maintain the stability and integrity. rRNAs confer the enzymatic activity (are ribozymes).
What are the 2 mechanisms for the initiation of translation?
1) Cap-dependant initiation- the initiation complex interacts with the 5’ cap and then scans in the 5’-3’ direction until the start AUG codon is found.
2) Internal ribosome entry site (IRES)- the initiation complex binds upstream of the initiation codon (starts in 5’ UTR opposed to at the cap).
Describe the main steps of cap-dependant initiation of translation.
1) eIF4E recognises and binds directly to the cap when signalled from growth factors
2) eIF4G (scaffold protein) then binds to eIF4E, and has domains where eIF4A and eIF4B can bind
3) PABP (poly-A binding protein) binds to eIF4G and causes the mRNA to circularise
4) eIF1A helps to generate a pool of free 40S subunits, and eIF2 binds the tRNA with methionine and this then binds the small subunit, forming the 43S complex
5) The 43S complex binds to the RNA and eIF3 within it binds the eIF4G
6) Scanning of the 5’ UTR occurs and the AUG codon within the Kozak sequence is found
7) At the start codon the 6OS subunit needs to bind the RNA, and eIF5 within the 40S subunit induces GTP hydrolysis when this binding happens and so acts as a checkpoint for correct 80A assembly
Describe the elongation cycle of eukaryotic protein synthesis.
1) Aminoacyl-tRNA binding- EF-1A in its GTP bound form will bring the next aminoacyl-tRNA to the A site by binding here
2) Peptidyl transfer- a peptide bond forms between the amino acid on the tRNA in the A site and the growing polypeptide chain in the P site. This moves the growing polypeptide from the P to the A site and elongates the chain by 1 amino acid
3) Translocation- EF-2-GTP hydrolyses to EF-2-GDP, which provides the energy for the translocation of the polypeptide from the A to the P site, kicking out the empty tRNA and allowing the elongation cycle to continue
How is transcription terminated?
eRF1 structurally mimics tRNA bound to EF-1A-GTP and so fits into the ribosomal A site. Here it recognises the stop codon and releases the completed polypeptide by catalysing a nucleophilic attack on the ester bond between the peptide and the P-site tRNA. This activity is stimulated by eRF3-GTP.
How much energy does translation require?
A total of 1ATP and 3GTP are required per cycle. 1ATP for initiation, 2GTP for elongation, and 1GTP for termination.
What elements can influence the translation of mRNA?
- Cap structure- if decap then mRNA is degraded
- PolyA tail- can be deadenylated
- Length of 5’ UTRS- lots of sites where regulatory proteins can bind
- Secondary structures close to 5’ end e.g. hairpins- need to linearise so complexes can bind
- IRES for cap-independent translation
- Short ORFs in front of main ORF control that are in right position for start of translation
- Binding sites for miRNAs- block translation and so regulate gene expression
What is nonsense mediated degradation and how does it occur?
It is degradation of mRNA molecules that have premature stop codons.
1) UPF proteins bind to the large subunit of the ribosome when the mutation is sensed, and instruct the ribosome that it is not the correct stop codon
2) Decapping enzyme is activated and cap removed
3) Exonuclease degrades the mRNA
What is non-stop mediated decay and how does it occur?
This is the type of mRNA decay required when the mRNA has no stop codon. Ski7 protein binds and interacts with the exosome, and causes degradation from the 3’ end. Degradation occurs by a Lys specific protease; everything is broken down and reused by the cell.
What happens to protein translation upon viral infection?
Viral infection induces the production of inteferons that bind to cell surface receptors and activate transcription of antiviral genes, and 2 key ones down regulate translation: RNase L (degrades RNA) and PRK (phosphorylates eIF2a, inhibiting translation initiation as it binds to eIF2b with a tight interaction so cannot translocate polypeptide from A to P site.
Describe the translation of picornovirus mRNAS by IRES-mediated mechanisms.
In infected cells eIF4G is cleaved which inactivates translation, however not the translation of picornovirus mRNAs as the 40S subunit binds IRES ( has no 5’ caps). This IRES mediated translation depends only on the C-terminal fragment of eIF4G to recruit the 40S subunit through the interaction with eIF3, so the cleavage only inhibits translation of host mRNA and not the virus mRNA.
