7 Protein Translation And Post-Translational Modification

Question 1

Q: Describe structure of a typical mRNA. Use a diagram.

Answer 1

A:
7MeG————–|————————–|———–AAAAAAn
5’ cap 5’UTR 3’UTR poly A

RNA is always written as 5’ on the left and 3’ on the right.

The 5’ cap is a modified base which is 7-Methyl Guanosine and it is the entry site for ribosome. (7 methyl guanylate cap)

Next to the cap, at the 5’ end, is a non-coding region known as 5’ UTR (untranslated region).

Then you get the coding region.

Followed by the 3’ UTR.

At the 3’ end you get a polyA tail

Question 2

Q: What are stop codons? (3)

Answer 2

A: UAA, UAG, UGA

Question 3

Q: What is the codon for methionine? Describe it.

Answer 3

A: AUG (essential aa- first amino acid of virtually every protein that is translated in the cells)

Question 4

Q: How is mRNA read? (compared to the direction that protein is synthesises)

Answer 4

A: Ribosome scans from the 5’ end (7MeG cap) of the mRNA.

Protein translation occurs in a 5’ to 3’ direction and the protein is synthesised in an N to C direction

Question 5

Q: Where does translation start and stop?

Answer 5

A: Translation starts at the first AUG (Met) and continues in frame.

Translation stops at the first IN FRAME stop codon.

Question 6

Q: Describe ribosomes and how do they differ in bacteria and eukaryotes? Similarity?

Answer 6

A: Bacterial ribosome is less complex than eukaryotic one - it has fewer proteins and fewer RNA.

2 Subunits

Question 7

Q: What is tRNA? Relation to amino acids? Structure? Enzyme?

Answer 7

A: The transporter of amino acids to the ribosome.

At least 1 tRNA per amino acid

Amino Acid is attached to the 3’ end- Amino Acid gets stuck onto the correct tRNA using aminoacyl tRNA synthetases.

Question 8

Q: Why are aminoacyl tRNA synthetases important? (2)

Answer 8

A: They are important in the fidelity of translation - they make sure that the correct amino acid is bound on to the correct tRNA. since there is one aminoacyl tRNA synthetase per amino acid.

Question 9

Q: Explain the mechanism by which aminoacyl tRNA synthetases work. Relation to diseases? (4)

Answer 9

A: Using the energy from ATP, the enzyme forms an intermediate known as an adenylated amino acid. The intermediate has the enzyme bound to AMP (derived from ATP which loses a pyrophosphate molecule) and the AMP is bound to the correct amino acid.

The adenylated amino acid then binds an appropriate tRNA. The AMP comes off and the enzyme dissociates.

The amino acid is transferred to the 3’ hydroxyl of the tRNA.

The enzymes can be mutated in many diseases such as cancers, neuropathies, autoimmune disease and metabolic disease

Question 10

Q: What are the 3 stages of translation?

Answer 10

A: 1. Initiation:

2. Elongation:
3. Termination:

Question 11

Q: How many ribosomes translate a piece of mRNA? Why?

Answer 11

A: -not by one ribosome at a time
-several ribosomes (polyribosomes) on the mRNA at one time - like a string of beads

much more protein can be produced in the same amount of time.

Question 12

Q: What are antibiotics and how do they work? Exploit?

Answer 12

A: natural products of bacteria and fungi that give them a selective advantage over other microbes.
by inhibiting protein synthesis.

Antibiotics exploit the differences between prokaryotes and eukaryotes.

Question 13

Q: Name examples of antibiotics. Include their mode of action. (5)

Answer 13

A: Streptomycin - Inhibits initiation
Tetracycline - Inhibits aa-tRNA binding
Erythromycin - Inhibits translocation
Chloramphenicol - Inhibits Peptidyl Transferase
Puromycin - Terminates Elongation Prematurely

Question 14

Q: Where does protein synthesis take place? (2)

Answer 14

A: Not in any organelle, in the cytoplasm (can occur in mito)

Question 15

Q: What type of proteins are synthesised in the rER? (2) location? What do they contain? where? describe.

Answer 15

A: Secretory and Transmembrane Proteins- type of amino acids that would like to sit within the lipid bilayer

Proteins that are destined to be secretory or transmembrane have a special sequence (first 20-24 amino acids) called a SIGNAL SEQUENCE.

The signal sequence is present at the N-terminus of the polypeptide (first sequence that is synthesised).

The signal sequence is rich in HYDROPHOBIC AMINO ACIDS

Question 16

Q: Describe the 4 steps involved in protein synthesis of secretory and transmembrane proteins (rER).

Answer 16

A: 1. The signal sequence (specific to secretory and transmembrane proteins) is recognised by a protein-RNA complex known as the Signal-Recognition Particle (SRP) -> SRP binds to the signal sequence and halts translation and

2. SRP binds to the SRP Receptor on the membrane of the rER = translation resumes.
3. As the protein continues to be translated, the bound SRP triggers the assembly of a protein channel within the membrane of the rER. The growing polypeptide chain is threaded through the channel, across the membrane and into the lumen of the rER. (If the protein is destined to be a transmembrane protein they will have an extra hydrophobic sequence to hold them in the membrane)
4. Once the signal sequence has done its job of getting the protein in to the rER, it is chopped off (cleaved) by signal peptidase and it’s degraded. You end up with the main protein which is folded.

Question 17

Q: List examples of post translational modification of proteins. (6) What do PTMs do?

Answer 17

A: increase protein diversity

Disulphide bond formation
Proteolytic cleavage
Glycosylation (addition of carbohydrate)
Phosphorylation (addition of phosphate)
Prenylation, Acylation (addition of lipid groups)
Hydroxylation

Question 18

Q: Where is insulin synthesised? What does it have? Use insulin as an example of PTM. (3) Result?

Answer 18

A: synthesised on membrane bound ribosomes on rER. It has a N terminus signal sequence which binds to SRP and allows it to be transferred across the membrane of the ER

Post-Translational Modification of Insulin:

• Signal sequence is removed and degraded
• Disulphide bonds form between cysteine residues within the polypeptide chain
• Before it is packaged into secretory vesicles, it is proteolytically cleaved in two positions to release the C chain

= leaves you with active, fully-functional insulin which consists of the A and B chains held together by the three disulphide bonds

Question 19

Q: Role of polyA tail?

Answer 19

A: protects the 3’ end from degradation

Question 20

Q: Describe initiation as a process in translation. (5)

Answer 20

A: pre-initiation complex consisting of eIF-2 + GTP + 40S + Met-tRNA binds to 7-MeG cap at 5’ end (because eIF4E and G have bound to the cap)

Preinitiation complex moves along mRNA until it reaches the first AUG

GTP hydrolyses to GDP which causes conformational change in preinitiation complex

allowing 60S subunit to bind to the complex

eIF-2 and GDP dissociate.

Question 21

Q: Describe elongation as a process in translation. (4)

Answer 21

A: Another tRNA binds to the A site of the ribosome

and peptidyl transferase catalyses the formation of the peptide bond between the two amino acids

First tRNA dissociates and the ribosome moves along

Elongation Factors promote the movement of the ribosome by using energy from GTP.

Question 22

Q: Describe termination as a process in translation. (3)

Answer 22

A: When the A site moves over a stop codon, a Release Factor binds

Peptidyl transferase then catalyses the transfer of the complete polypeptide chain to water and releases it from the ribosome.

Everything then dissociates.