Translation and Protein synthesis.2

Question 1

Compare protein synthesis systems in eukaryotic vs prokaryotic cells.

Answer 1

EUK: Transcription starts in the nucleus, mRNA transported into cytosol and translated here. Monocistronic mRNA generated.

PROK: Transcription and translation occur simultaneously to save resources and time. Polycistronic mRNA generated.

Question 2

What is the 5’ cap found on mRNA?

Answer 2

Guanine nucleotide cap at the 5’ end… essential for the ribosome to bind to the 5’ end of the mRNA.

Question 3

What is the 3’ Poly(A) tail?

Answer 3

50-250 adenine nucleotides, which stabilise the mRNA and play an important role in transcription termination.

Question 4

Where does initiation usually occur?

Answer 4

At the first AUG closest to the 5’ end of an mRNA.

Question 5

What are the two main mechanisms for the initiation of translation?

Answer 5

Cap-dependent initiation:
- Initiation complex interacts with the 5’ cap structure and scans in a 5’-3- direction until the start AUG codon.

IRES (internal ribosome entry site):

• I.e. cap independent.
• Initiation complex binds upstream of initiation codon.

Question 6

What are eukaryotic initiation factors?

Answer 6

Proteins in eukaryotes that mediate the initiation of translation.

Question 7

Outline the steps involved in the initiation of translation.

Answer 7

• Circularisation of mRNA.
• Formation of the 43S complex and initiation of translation
• Binding of the 43S ternary complex and scanning of the 5’UTR for the start codon
• Assembly of the 80S ribosome complex.

Question 8

Outline the process of mRNA circularisation.

Answer 8

eIF4 complex associates with the 5’ and 3’ ends of the mRNA… it contains various subunits:

• eIF4G… will associate to the PABP bound to the polyA tail.
• eIF4E… binds the 5’ cap structure.
• eIF4A and eIF4B can bind to the complex as scaffold created where they can bind… eIF4A has helicase activity… ATP dependent RNA helicase… unwinds the secondary structure in 5’ untranslated region of mRNA. eIF4B stimulates this helicase activity.

Question 9

What is the 43 pre-initiation complex?

Answer 9

A ribonucleoprotein complex that exists during an early step of eukaryotic translation initiation.

Question 10

What is the purpose of the 43S complex?

Answer 10

To load the first Methionine start codon amino acid on to the chain to allow translation to commence, and begins the assembly of the 80S ribosome.

Question 11

What components make up the 43S PIC?

Answer 11

It is a preinitiation complex (PIC) that contains the small ribosomal subunit (40S), bound by the initiation factors eIF1, eIF1A, eIF3 and the eIF2-Met-tRNA.met-GTP ternary complex.

Question 12

How is the 43S complex assembled?

Answer 12

eIF1A… helps to generate a pool of free 40S subunits.

eIF1A and eIF3 promote the binding of the ternary complex to the 40S subunit.

eIF2 binds the methionine amino acid… the ternary complex has the affinity for the 40S subunit… where the mRNA sits.

Together this forms the 43S complex.

Question 13

Outline the process of the binding of the 43S ternary complex and scanning of the 5’UTR for the start codon.

Answer 13

Once mRNA has circularised, get the binding of the 43S ternary complex to the cap site.

Once in place, begins scanning for the Kozak sequence… locates the place where translation should stat and first amino acid goes.

Question 14

What is the importance of eFI3 in the 43S binding?

Answer 14

It is an essential component in recognising the initiation complex of the mRNA.

Question 15

How is the 80S ribosome complex assembled?

Answer 15

60S subunit binds to the start site… mainly regulated through eIF5.

Question 16

Outline what happens in the elongation phase of translation.

Answer 16

Polymerisation reaction, requiring two main elongation factor proteins (EF1 and EF2)… cycle of GTP-ase activity.

EF1a.GTP is active… recognised by the loaded tRNA… the energy from the GTP will enable the loaded aminoacyl tRNA to bind to the A site of the 60S subunit… this inactivates the EF1a.GTP as GTP is hydrolysed to GDP. Can be reactivated by EF-1B which will swap the GDP for GTP.

Then peptidyl transfer occurs where the growing polypeptide chain transferred from P site to new tRNA on the A site… EF2.GTP enters ribosome, pushing the new tRNA with polypeptide chain on it into the P-site, and deacylated first tRNA into the exit (E) site (only in its GTP state)… the tRNA in the E site will be ejected in the next cycle. This process frees up the A site, so another aminoacyl tRNA can come and join.

When EF2.GTP is hydrolysed… EF2.GDP leaves the ribosome… an cycle is repeated.

Question 17

What factors are involved in termination of translation?

Answer 17

Releasing factors (eRFs).

Question 18

Outline the process of termination of translation.

Answer 18

eRF1 structurally mimics tRNA that is bound to EF1a.GTP.

eRF1 fits into the ribosomal A site, where it recognises the stop codon.

It then releases the completed polypeptide by catalysing a nucleophilic attack on the ester bond between eh peptide and the P-site tRNA.

The catalytic activity of eRF1 is stimulated by the GTP-bound form of another releasing factor eRF3.

Question 19

Compare the structure of tRNA and eRF1.

Answer 19

The overall shape and structure of the eRF1 resembles tRNA structure.

Domains 1, 2 and 3 of the eRF1 correspond to the anticodon loop, aminoacyl acceptor stem and T-stem of the tRNA molecule.

Therefore, when no more codons, releasing factor acts as signal to show polypeptide has been fully translated and must be released from stop site.

Question 20

How does proofreading occur in translation?

Answer 20

Mediated with affinity of interaction.

Higher rate of dissociation of the ternary complex if there is incorrect base-pairing.

This dissociation of incorrect ternary complexes takes place prior to GTP hydrolysis so the aminoacyl tRNA from ternary complex does not remain bound to the ribosome.

Question 21

List 6 mechanisms that can regulate protein translation.

Answer 21

Extracellular stimuli and stresses, signal transduction pathways, energy status, nutrient availability, cellular metabolites and miRNA.

Question 22

Why is it important to regulate translation?

Answer 22

Because it is very costly in energy, to produce proteins when and where they’re needed, produce proteins locally i.e. synapse.

Question 23

What is the difference between general protein synthesis and specific protein synthesis.

Answer 23

General protein synthesis:
-I.e. need lots of proteins for growth, proliferation, cell division, increase in mass etc. … can produce the cytoskeleton, membranes, amplification of many proteins etc.

Specific protein synthesis:
-Allows for the translation of specific mRNAs… for quick and specialised regulatory responses. i.e. under stressed conditions, etc. produces the proteins it needs quickly, for a quick and specialized response.

Question 24

Name two signaling pathways that regulate the initiation of translation.

Answer 24

mTOR and MAPK.

Question 25

What is mTOR?

Answer 25

A cytosolic protein kinase mainly found on lysosomes (serine/ threonine kinase).

It is about 2500 amino acids, and known as the lazy kinase as just waits for the substrate to be brought to it.

Question 26

What factors does mTOR respond to?

Answer 26

Nutrients (amino acids and glucose), mitogens and growth factors, cellular energy (ATP and metabolites.).

Question 27

What processes does mTOR control?

Answer 27

Ribosomal biogenesis, amino acid transport and initiation of translation (once there is enough ribosomes and amino acids).

Question 28

What does mTOR signalling control?

Answer 28

Protein biosynthesis, amino acid uptake, autophagy, glucose uptake, transcription, FA oxidation, mitochondrial biogenesis, glycolysis.

Question 29

What can dysregulation of the mTOR signaling pathways lead to in terms of human pathologies?

Answer 29

Cardiac hypertrophy, inflammation ,cancer, neurodegeneration, metabolic disorders.

Question 30

What are the two regulatory mTOR complexes that form in cells?

Answer 30

These complexes have different substrates and respond to different factors.

mTOR complex 1: Raptor.

mTOR complex 2: Rictor.

Question 31

What are the characteristics of Raptor?

Answer 31

• Mainly responds to nutrients and AMP/ATP.
• Phosphorylates 4E-BP1 and S6K…
• Engaged in cell growth and metabolism.

Question 32

What are the characteristics of Rictor?

Answer 32

• Mainly responds to hormones and growth factors.
• Phosphorylates a serine/ threonine kinase.
• Engaged in cytoskeletal rearrangements and in proliferation.

Question 33

What is eIF4E-BP and what is the effect if this protein is phosphorylated by mTOR?

Answer 33

These are translational inhibitors.

This is a protein that competes with eIF4G for binding to eIF4E (the cap binding protein)… when bound… hold the eiF4E tightly, so o longer available to bind to the cap… no circularisation… inhibits translation.

If mTOR phosphorylates this protein upon receiving signal from the cell that translation is needed… the eIF4E binding is weakened and so it is released, and can now interact with the eIF4G and promote translation.

Question 34

What is the function of S6K1?

Answer 34

It is a protein kinase, phosphorylating serines and threonines.

It can phosphorylate eIF4B, so is a positive regulator of the cap binding eIF4F complex.

It can also phosphorylate and promote the degradation of pDCD4 which is an inhibitor of eIF4B.

Question 35

What role does the MAPK pathway play in translational control?

Answer 35

It phosphorylates the MNK1 protein… which would phosphorylate eiF4E specifically… so that is would dissociate from the 4E-BP inhibitor and bind to the eIF4G and cap.

Question 36

What are small regulatory RNAs?

Answer 36

21-28 nucleotides in length, and regulate gene expression in animals and plants.

Question 37

What is the function of siRNAs?

Answer 37

Guide mRNA cleavage.

Question 38

What is the function of miRNAs?

Answer 38

miRNAs inhibit translation in humans and regulate around 30% of human genes.

Question 39

Describe miRNA expression.

Answer 39

It is tissue/ developmental stage specific.

miRNA profiles can be altered in human disease.

Question 40

What is the role of miRNAs in the regulation of translation?

Answer 40

They bind to the 3’ UTR and form a specific complex which would stall the process of translation… inhibit translation.

Question 41

What 7 factors influence translation of mRNA?

Answer 41

-Cap structure at the 5’ end… decapping can halt translation.
-Poly(A) tail at the 3’ end… de-adenylation can aldo halt translation.
-Length of the 5’UTRs.
-Secondary structures close to the 5’ end.
-IRES: Ribosome entry site (mediated cap-independent translation).
-Short ORFs in front of the main ORF.
Binding sites for trans-acting regulatory factors (proteins, miRNA etc).

Question 42

What are exon junction complexes?

Answer 42

The location where the regulatory proteins will bind.

Question 43

What happens when there are no more exon junction complexes?

Answer 43

The ribosome knows it must stop translation.

Question 44

What happens if there is a mutation coding for a premature stop codon?

Answer 44

• I.e. Stop codon produced in front of the exon-exon junction.
• Mechanism would recognise that this is not the real stop codon… as it should be behind the exon-exon junction… and so would continue to the next junction:
  …Binding of upstream factors (Upf) proteins.
  …Activation of the decapping enzymes, to remove the cap.
  …The involvement of exonuclease.
  …To remove the faulty RNA.
  …Degradation of mRNA.

Question 45

What happens if there is no stop codon at all?

Answer 45

• Ribosome continues to the poly A tail… lysine in the polypeptide chain… problem as proteins don’t usually have sequence of multiple lysines in their structure.
• Ski7 would recognise this and bring together exosome.
• The exosome would engage the RNA RNases… would digest RNA from 3’ to 5’ and lysine specific proteases, to remove the nuisance amino acids.
• I.e. degradation of mRNA without a stop codon.
• Protein degradation by Lys specific protease.

Question 46

How does the body respond to viral infection with inhibition of general translation?

Answer 46

-Viral infection induces the production of interferons as part of the rapid innate immune response.
-Interferons bind to the cell surface receptors and activate the transcription of antiviral genes.
-Two interferon-induced genes are key players in downregulation of translation:
…RNase L (degrade RNA).
…PKR (protein kinase RNA-activated)…PKR phosphorylates eIF2a during the infection of cells, inhibiting translation initiation.
-Because provide high affinity binding site for the exchange factor… binds so tightly that it will not exchange… bound in GDP state, so don’t have exchange factor… blocking translation.

Question 47

Outline the mechanism of translation of picornavirus mRNAs.

Answer 47

• Has no 5’ cap structure in mRNAs of picornoviruses (during viral infection, 40S subunit binds internal ribosome entry site (IRES)).
• In infected cells, eIF4G is cleaved, inactivating translation of cellular mRNAs.
• The IRES-mediated translation does not require the presence of a cap structure but depends on the C-terminal fragment of eIF4G to recruit the 40S subunit through interaction with eIF3.

Question 48

Outline the mechanism of translation of Hepatitis C mRNAs.

Answer 48

Translation only requires the participation of eIF3… which mediates the interaction between the biral IRES and the 40S ribosomal subunit… don’t require any other initiation factors for the formation of the initiation complex.
Dramatic change in conformation of the 40S subunit when it binds Hep C virus IRES sitting at the AUG in the P site.

Question 49

How does Puromycin inhibit bacterial translation?

Answer 49

Aminoacyl-tRNA analog… causing premature chain termination.

Question 50

How does Tetracycline inhibit bacterial translation?

Answer 50

Inhibits aminoacyl-tRNA binding to the A site.

Question 51

How does Chloramphenicol inhibit bacterial translation?

Answer 51

Inhibits peptidyl transferase activity.

Question 52

How does Erythromycin inhibit bacterial translation?

Answer 52

Binds peptidyl transferase and blocks translocation.