Protein Synthesis Flashcards
How does the 3’ poly A tail of mRNA contribute to its stability?
The 7-methyl guanosine cap?
What are they both used for?
What does the 3’ end of the tRNA have?
What is the anticodon loop responsible for in aminoacyl-tRNA complex?
How many tRNAs are there for 1 aa?
How many aminoacyl-tRNA synthetases are there for an aminoacyl-tRNA?
What does aminoacyl-tRNA synthetase do?
What are the two domains of aminoacyl-tRNA synthetase?
What are the two sites responsible in the catalytic domain?
Protection of degradation from endonucleases
Protection of degradation by RNases
3’A: transcription termination, translation & exporting mRNA out of nucleus
5’: recognition by ribosome for translation
CCA sequence to attach amino acid
Specificity
Several
1 - only 20 total
Catalyse formation of ester bond between amino group & tRNA to bring amino acid into its active form (aminoacyl-tRNA) for peptide bond formation
Anticodon-binding & catalytic
Activation: of amino acid - catalyses aminoacyl-adenylation & tRNA aminoacylation
Editing: proofreads & hydrolyses misactivated amino acid/tRNA
What are ribosomes made up of? What do each element do?
What is the rate limiting step of translation initiation?
What is the IRES (internal ribosome entry site) translation initiation in short?
What does 5’cap dependent initiation rely on?
ribosomal proteins - stability & integrity
rRNA - ribozyme for catalysis
Hydrolysis of ATP/GTP and formation of complex of ribosome, mRNA & methionine-tRNA initiator
40S subunit binds to IRES (RNA) to form ribosome
circularisation of mRNA
what are the 3 steps in 5’ cap dependent initiation for circularisation of mRNA?
What are the 4 reasons why circularisation is important?
What helps generate a pool of 40S subunits?
What promotes binding of 43SPIC (complex of eIF) to the 40S?
What kind of complex is 43S PIC?
- eIF (eukaryotic initiation factor) interacts with 5’ cap on mRNA
- interaction changes the eIF conformation, allowing for more factors (scaffold proteins) to bind into a complex
- eIF4G complex interaction with Poly(A) binding protein allows for binding to the poly(A) tail of mRNA = circularisation (& formation of 43S PIC)
increases local concentration of initiation factors on mRNA
2. efficient protein synthesis can occur
3. proof-reading function: ensures only intact/stable mRNA with 5’ cap can be translated
4. allows interaction between events happening at 5’ and 3’
eIF1A
eIF1A & eIF3
Ternary complex
in 3 steps, how is the 80S ribosome assembled?
What is the difference between the Kozak sequence & Shine Dalgarno sequence?
- 40S subunit binds upstream of the AUG start codon & scanning begins
- GTP hydrolysis induced by eIF5 & binding of 60S subunit is checkpoint assembly of 80S ribosome
- ribosome scans along mRNA until finds specific start AUG codon (Kozak sequence in eukaryotes)
Kozak sequence is ribosome-binding site containing the AUG start codon (eukaryotes)
Shine dalgarno ribosome binding site is upstream of the start AUG codon (prokaryotes)
What are the 3 steps of translation elongation?
How does proofreading of translation happen?
What is this due to?
When does proofreading happen?
- Eukaryotic elongation factor eEF2-GTP (GTP bound) enters ribosome & pushes tRNA into P site (from A site) & first tRNA (deacetylated) into E (exit) site
- GTP is hydrolysed in this process & eEF2-GDP leaves ribosome
- cycle repeats
If base pairing between codon & anticodon isn’t perfect, aminoacyl-tRNA doesn’t bind to the ribosome (occurs in A site & doesn’t move to P site)
Imperfect binding means aminoacyl tRNA has low affinity for the ribosome so it will not bind
Before GTP hydrolysis
In 3 steps, how is translation terminated?
How is eRF1’s catalytic activity stimulated?
What do the 3 domains of eRF1 mimic in the aminoacyl-tRNA structure?
- eRF1 (eukaryotic release factor) fits into ribosome A site as mimics tRNA bound in eEF1a-GTP complex
- eRF1 enters A site & recognises stop codon
- Completed polypeptide is released by catalysing nucleophilic attack on ester bond between peptide & P-site tRNA
by eRF3-GTP
domain 1 mimics anticodon loop - recognises stop codons & specifically binds
domain 2 mimics aminoacyl acceptor stem
domain 3 mimics T-stem of tRNA
Why does protein synthesis need to be regulated? (3)
What is proteostasis?
What is mTOR?
What 2 complexes are formed in response to nutrients/growth factors in the cell?
How are the 2 complexes different?
- produce protein when cell requires it
- produce proteins locally (synapses)
- produce single proteins in specialised manner
Dynamic balancing of translation regulation, folding, post-translational modifications, degradation - unbalancing leads to pathological change & disease
mammalian target of rapamysin
mTORC1 & mTORC2
C1 has Raptor (regulatory associated protein of mTOR) and rheb (GTPase for activation of complex) whereas C2 has rictor (rapamysin-insensitive companion of mTOR)
What does the activation of mTORC1 lead to in high nutrients, stresses etc?
What else does mTORC1 activate?
How does this cause a knock on effect with cAMP?
Phosphorylates 4E-BP1 allowing for eIF4E to unbind for translation initiation
Protein kinase B
Phosphorylates adenylyl cyclase (activation) to convert cAMP to ATP
How are MINK kinases activated?
What does its activation lead to?
What happens with IRP (iron regulatory protein) binds to mRNA specifically?
What happens when maskin binds to eIF4E?
Phosphorylation from p38MAPK & Erk1/2
Phosphorylation of eIF4E- allowing it to unbind from MINK so can associate with eIF4G for protein synthesis/bind to 5’ cap
Allows for formation of eIF4E complex to activate translation
Inhibits translation - prevents function
How is cyclin B1 translated during the M phase?
What happens in the S phase?
How do miRNAs inhibit translation/silence RNA?
PABP dislodges maskin from eIF4E allowing for its translation through circularisation of mRNA
Maskin binds to eIF4E preventing mRNA from circularising & so inhibits translation intiation
Base pair to complimentary mRNA at 3’ end - destabilises the mRNA so degraded by endonucleases
What are exon-junction complexes?
What is their function?
Nonsense mediated degradation is when the ribosome encounters a pre-mature stop codon on the mRNA. What are the 3 steps for degradation?
Non-stop mediated decay occurs when there is no stop-codon on the mRNA. what are the 3 steps for mRNA and polypeptide degradation?
What can disregulation of protein translation lead to?
Formed at re-joining of exons in mRNA after introns spliced out
Instruct ribosome to continue with translation as drive ribosome to continue
- Upf proteins bind to ribosome (that has stopped at premature stop codon)
- Decapping enzyme is activated & uncaps 5’ of mRNA (destabilisation)
- Exonuclease recognises uncapped 5’ end of mRNA & degrades it
When the ribosome reaches the poly(A) tail (due to no stop codon), Ski7 protein binds to the ribsome
2. Exosome interacts with the Ski7, resulting in the dissociation of the ribosome-ski7 complex & degradation of the mRNA
3. The completed polypeptide is degraded by Lys specific protease
Cancer, metabolic disorders, viral infection
What is induced when a virus infects a host?
How do they induce activation of transcription of antiviral genes (to downregulate translation of viral proteins)?
What are the 2 interferon-induced genes and what do they do?
Why are these genes important for most viral mRNA?
Picornavirus mRNA doesn’t have a 5’ cap on the mRNA - how is translation inactivated?
Hepatitis C mRNA?
How does coronvavirus mRNA inhibit host cellular mRNA translation?
Interferons
Bind to cell-surface receptors
RNase L (degrades RNA)
PKR (protein kinase RNA-activated) - phosphorylates eIF2a resulting in inhibition of translation initiation
They have 5’ cap so target 5’ cap dependent translation
40S binds to IRES on viral RNA - in infected cells, eIF4G is cleaved - so inhibition of translation initiation (eIF4G required to recruit 40S)
40S subunit changes conformation when binds to hepatitis C IRES on mRNA - so cannot bind to 60S
activates stress inducing pathways (preventing eIF4E unbinding) - affects stability of cellular mRNA allowing viruses to hijack host cell machinery
How does phosphorylation of a protein occur?
What is released?
What amino acids side chains are most modified and why?
What do phosphotases do?
What is transphosphorylation?
What is the MAPK pathway?
Phosphate added to OH side chain through protein kinase & ATP
Pi & ADP
Serine, threonine, tyrosine - all have OH
Removes phosphate from protein
Ligand-induced dimerisation allows for kinase domains to phosphorylate neighbouring ones
MAPKKK -> MAPKK -> MAPK -> transcription factors
What is the inactive state of c-SRC ?
How is the active state of c-SRC induced?
What does activation of this pathway promote?
How is FOXO’s function regulated by phosphorylation?
What are protein kinases involved in in drug discovery?
Phosphorylated SH1 kinase is inactivated by SH2 domain binding to it
Nutrients, growth factors etc inducing phosphorylation of tyrosine419 - binds to SH2 with higher affinity changing conformation of SH1 kinase into its active state
Cell proliferation & cancers
Phosphorylation allows for its translocation from cytoplasm to nucleus to act as a transcription factor
Drug discovery programs target them as they can be mutated/activated & induce tumour formation
How are histones acetylated?
Where do acetyl groups bind?
What does acetylation of histones do?
How are histones de-acetylated?
What domains recognise/associate to acetyl groups on tails?
What does histone methylation result in?
What is an activator of gene transcription?
What about inactivator? What happens?
What are the chromo/bromo domains essential for?
Acetyl-coA & HAT (histone acetyl-transferase)
N-terminal lysine tails of histones
Histone association with chromatin to relax allowing transcription factors access DNA (euchromatin)
H2O & HDAC (histone deacetylase) into acetate
Bromodomain
Transcription activation/deactivation depending on the N-terminal tail
Methylation of lysine 4 histone 3
Methylation of lysine 9 histone 3- results in chromodomain binding to methyl group & produces tight interaction between chromatin & histone (heterochromatin)
Protein-protein interaction
