NAGE 6

Question 1

In translation, what is the entry point for a ribosome?

Answer 1

The 5’ cap.

Question 2

What is present next to the 5’ cap?

Answer 2

Non-coding region called 5’ UTR (untranslated region).

Question 3

Describe the sequence of an mRNA strand.

Answer 3

5’ cap - 5’ UTR - coding region - 3’ UTR - polyA

Question 4

What does the poly-A tail do?

Answer 4

Protect the 3’ from degradation.

Question 5

Why is methionine important?

Answer 5

Translation starts at the first AUG (met).

Question 6

When does translation stop?

Answer 6

At the first in frame stop codon.

Question 7

Why are tRNAs important?

Answer 7

They transport aa to ribosome.

Question 8

Where is the amino acid attached in tRNA.

Answer 8

On the 3’ end.

Question 9

How are aa attached onto the correct tRNA?

Answer 9

Using aminoacyl tRNA synthetases.

Aminoacyl tRNA synthetases are important in ensuring that the correct aa is bound to the correct tRNA.

Question 10

How do aminoacyl tRNA synthetases work?

Answer 10

1. Using ATP hydrolysis for energy, the enzyme forms intermediate - Adenylated AA.
2. Adenylated AA consists of Enzymes attached to AMP attached to correct amino acid.
3. Adenylated AA then moves to appropriate tRNA. AMP comes off and enzyme dissociates.
4. AA transferred to 3’ hydroxyl of tRNA.

Question 11

Explain the initiation step of translation.

Answer 11

1. 60s and 40s ribosome subunits dissociate in cytoplasm.
2. Pre-initiation complex forms. This consists of Met-tRNA, eIF2, GTP and a 40s unit bound together.
   (ONLY Met-tRNA CAN BIND TO 40S ALONE).

Other initiation factors bind to 5’ cap.

3. eIF4E and eIF4G bind to 7-MeG cap and is recognised by pre-initiation complex. Pre-initiation complex moves 5’ to 3’ until anticodon of Met-tRNA pairs up with AUG codon.
   When AUG is recognised, GTP on eIF2 is hydrolysed to give energy for base pairing.
   GTP hydrolysis gives conformational change in complex, allowing 60S to bind - full ribosome formed. Initiation factor (w/ bound GDP) dissociates.

Question 12

What is the site that the Met-tRNA binds to called?

Answer 12

The p site.

P site on the left and A site on the right.

Question 13

Describe how Elongation works.

Answer 13

1. New tRNA binds to adjacent A site on ribosome next to initiator Met.
2. Peptide bond formed between two amino acids. Reaction catalysed by peptidyl transferase (located on 60s subunit).
3. Ribosome slides along. Initiator tRNA falls off, tRNA (2) is now at P site. Whole process facilitated by Elongation Factors, which use GTP energy to promote movement of ribosome along.
4. New tRNA occupies A site. Process repeats. EFs use pauses that allow incorrect base pairs to dissociate.

Question 14

Explain how termination works.

Answer 14

1. Ribosome moves to a stop codon. Vacant A site is over the stop codon. Release Factor proteins bind to empty A site (THESE ARE NOT tRNAs).
2. Release factors bind to stop codon. Peptidyl transferase catalyses transfer of completed polypeptide chain to water. Released from ribosome. EVERYTHING DISSOCIATES.

Question 15

Where does protein synthesis occur?

Answer 15

Cytoplasm

Question 16

What is the special sequence that secretory or transmembrane proteins have?

Answer 16

Signal sequence (first 20-24 amino acids).

Signal sequence present at N-terminus of polypeptide (first sequence that is synthesised).

Signal sequence rich in Hydrophobic amino acids.

Question 17

How are secreted/transmembrane proteins formed?

Answer 17

1. “Signal-Recognition Particle” (SRP - protein/RNA complex) recognises a signal sequence and binds to signal sequence. Translation halted.
2. SRP binds to SRP receptor on rER membrane.
3. Binding of SRP to SRP receptor continues translation.
4. As translation continues, SRP triggers assembly of protein channel on the membrane of rER. Growing polypeptide chain threaded through protein channel into rER lumen.
5. Once in lumen, IF protein destined to be a transmembrane protein, they have an extra hydrophobic sequence to hold them in membrane,
6. Once protein is in rER, the signal sequence is chopped off via signal peptidase. Main protein remains, which is folded.

Question 18

Examples of post translational modification?

Answer 18

1. Disulphide bond formation
2. Proteolytic cleavage
3. Glycolysation
4. Phosphorylation
5. Prenylation/acylation
6. Hydroxylation

Question 19

How is insulin post-translationally modified.

Answer 19

1. Signal sequence removed and degraded.
2. Disulphide bonds form between cysteine residues (3 DS bonds formed). Protein folds forming PROINSULIN.
3. Before packaging into secretory vesicles, proteolytically cleaved in two positions, giving the C chain.
4. Active, functional insulin remains, which A and B chains held together by the3 DS bonds.