Translation and post-translational modification

Question 1

Describe the structure of typical mRNA.

Answer 1

mRNA has a 7-methylguanylate cap at the 5’ end followed by a 5’ untranslated region (UTR) You then get the coding region in the middle At the end you get a 3’ UTR and the poly-A tail

Question 2

What enzyme is involved in the transfer of an amino acid to tRNA?

Answer 2

Aminoacyl tRNA synthetase

Question 3

What is the Methionine codon?

Answer 3

AUG

Question 4

What is the significance of Methionine?

Answer 4

It is the first amino acid in virtually all polypeptides

Question 5

What are the stop codons?

Answer 5

UAA, UGA, UAG

Question 6

How do tRNAs work?

Answer 6

• there is one tRNA per amino acid
• the amino acid gets stuck onto the correct correct tRNA using aminoacyl tRNA synthetases
• There is one aminoacyl tRNA synthetase per amino acid
• They are important in the fidelity of translation

Question 7

How does Aminoacyl tRNA synthetase work?

Answer 7

Aminoacyl tRNA synthetase gets activated (by ATP hydrolysis) to form adenylated amino acid which binds with a molecule of AMP and binds to the relevant amino acid This adenylated amino acid complex then binds to the tRNA and transfers the amino acid to the 3’ end of the tRNA The adenylated amino acid and AMP dissociate

Question 8

What are the three stages of translation?

Answer 8

Initiation – Elongation - Termination

Question 9

What components are make up the preinitiation complex?

Answer 9

40S ribosomal subunit - Methionine tRNA - eIF2 (initiation factor) - GTP

GTP powers the reaction

Question 10

Describe initiation.

Answer 10

The preinitiation complex recognises initiation factors (eIF4E and G) on the 7MeG cap and bind to the mRNA It moves along the mRNA in a 5’ to 3’ direction until it reaches the first in-frame AUG (methionine) Here the GTP is hydrolysed providing energy to ensure correct base pair matching The GDP and eIF2 then dissociate causing a conformational change, which allows the binding of the 60S ribosomal subunit to form a full ribosome

Question 11

Describe elongation.

Answer 11

The next charged tRNA comes and binds to the A site of ribosome Peptidyl transferase catalyses the formation of a peptide bond between the two amino acids The tRNA from the P site then dissociates and the ribosome moves along

Question 12

What proteins facilitate elongation?

Answer 12

Elongation factors

Question 13

Describe termination.

Answer 13

When the A site of the ribosome moves over the first in-frame stop codon, a release factor (a type of protein) binds instead of a tRNA The release factor transfers the growing polypeptide chain to water thus terminating the polypeptide and allowing it to detach from the ribosome

Question 14

How do lots of antibiotics work?

Answer 14

lots of antibiotics work by inhibiting protein synthesis

they exploit the differences between eukaryotes and prokaryotes

Question 15

where does protein synthesis actually take place?

Answer 15

it takes place in the cytoplasm as from here it is passed through membranes to enter the compartments

the only exception is some protein synthesis happens in the mitochondria

Question 16

how do you get proteins into the compartments?

Answer 16

• the secretary and transmembrane proteins are synthesised in the rough endoplasmic reticulum
• 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 rich in HYDROPHOBIC AMINO ACIDS

Question 17

Where is the signal sequence found and what does it consist of?

Answer 17

It is found at the N terminus It mainly consists of hydrophobic amino acids

Question 18

Describe how proteins enter the ER.

Answer 18

The signal sequence binds to a signal recognition particle (SRP) and this binding halts translation

The SRP then binds to an SRP receptor on the ER membrane and translation resumes

The binding of SRP to the SRP receptor triggers the assembly of a protein channel through which the polypeptide is threaded into the RER

Question 19

What extra feature do proteins that are destined to be transmembrane have?

Answer 19

They have an extra hydrophobic sequence to hold them in the membrane

Question 20

What happens once the protein has entered the compartment?

Answer 20

The signal sequence is cleaved by signal peptidase

• we end up with the main protein which is folded

Question 21

Describe, in full, the post translational modification involved in the production of insulin.

Answer 21

You begin with preproinsulin, which has an N-terminus signal sequence which allows it to move across the membrane into the ER lumen

Once it enters the ER the signal sequence is cleaved and it 3 disulphide bonds form between cysteine residues. The protein folds… this makes it proinsulin.

before it is packaged into the secretory vesicles, it is proteolytically cleaved into two positions to release C chains

this leaves us with fully functional insulin which consists of the A and B chains held together by disulfide bonds

Question 22

why modify proteins?

give examples of modifications

Answer 22

• modifying proteins increases the diversity of the proteins

examples include

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