10 - Ribosomes and Cotranslation

Question 1

What part of the ribosome is responsible for the translation reaction itself, and what are its components?

Answer 1

The peptidyl transferase centre is made up of the E, P and A sites

Question 2

Describe the subunit makeup of the prokaryotic ribosome.

Answer 2

A 70S complex made up of a 50S and a 30S subunit that bind over the mRNA and hence both contain parts of the peptidyl transferase centre.

Question 3

What is the total mass of 70S prokaryotic ribosome?

Answer 3

2.5MDa

Question 4

What is the relative proportion or rRNA to protein in the 70S ribosome?

Answer 4

65% rRNA

35% protein

Question 5

What have been the most useful tools for elucidating the ribosome structure?

Answer 5

Cryo-EM and XRD

Question 6

What is the molecular mass of the 30S subunit?

Answer 6

0.8MDa

Question 7

What is the molecular mass of the 50S subunit?

Answer 7

1.5MDa

Question 8

How many proteins are attached to the 30S ribosome?

Answer 8

21

Question 9

How many base pairs of rRNA are present within the 30S ribosome?

Answer 9

1.5Mbp

Question 10

What is the sedimentation coefficient of the 30S subunit after all the proteins have been removed?

Answer 10

16S

Question 11

What is the function of the 30S subunit?

Answer 11

It is the first to bind to the mRNA, which enters and exits the ribosome through it. It is hence responsible for recruitment and jointly responsible for the fidelity due to its large role in facilitating the m-tRNA interaction.

Question 12

How many rRNA chains are present in the 30S subunit?

Answer 12

1

Question 13

How many rRNA chains are present in the 50S subunit?

Answer 13

2

Question 14

How many proteins are attached to the 50S ribosome?

Answer 14

34

Question 15

What are the sedimentation coefficients of the isolated 50S subunit rRNA chains?

Answer 15

5S and 23S

Question 16

What is the total number of base pairs of rRNA present in the 50S subunit?

Answer 16

3Mbp

Question 17

What is the function of the 50S subunit?

Answer 17

Contains the peptidyl transferase centre, so is reponsible for catalysing peptide bond formation.
Also contains the peptide exit tunnel.

Question 18

What is the rate of peptide bond formation in a 70S ribosome?

Answer 18

20 per second.

Question 19

How long is the ribosomal exit tunnel?

Answer 19

100A

Question 20

What is the widest diameter of the ribosomal exit tunnel?

Answer 20

20A

Question 21

What rRNA and protein components make up the ribosomal exit tunnel?

Answer 21

23S subunit and proteins L4 and L22.

Question 22

What property of the ribosomal exit tunnel prevents interaction with the nascent peptide?

Answer 22

It is lined with hydrophilic residues.

Question 23

What delivers the tRNA to the active site?

Answer 23

EF-Tu GTP

Question 24

If the tRNA delivered has a matching anticodon to the mRNA codon, what happens?

Answer 24

This causes an rRNA conformation change, which spins the tRNA by 5°, but not yet into the A site.
There is also domain closure of the 30S subunit shifting the EF-Tu GTP into position.

Question 25

What happens when match identification causes the EF-Tu GTP to assume its active position?

Answer 25

The GTP is hydrolysed leading to a 100° change in conformation of the EF-Tu causing it to dissociate.
This also powers the entry of the tRNA into the A site.

Question 26

How are the arms of the tRNA molecules named?

Answer 26

Acceptor arm (binds AA)
Anticodon arm
C, D and T arms.

Question 27

How much structure forms in the ribosomal exit tunnel?

Answer 27

It is only wide enough to allow for some limited helical structure to develop.

Question 28

Which protein is a common model for cotranslational folding, and why?

Answer 28

Luciferase, as it is unable to fold under any other circumstances.

Question 29

How does luciferase fold?

Answer 29

Exclusively cotranslationally, as the N-terminal domain folds as it exits around which the rest of the protein folds.. Hence unlike RNaseA it would not refold if denatured.

Question 30

How can cotranslational folding be imaged?

Answer 30

By removing the stop codon from a gene in order so that it doesn’t recruit release factors and hence gets stuck in the ribosome.

Question 31

What protein production technique is often used to image ‘folding snapshots’ from cotranslational folding?

Answer 31

Cell-free expression, as this allows for easy purification.

Question 32

What imaging technique is used to view cotranslational folding?

Answer 32

NMR, as this allows continuous visualisation of the folding process as it, uhh… unfolds, as opposed to a still snapshot.

Question 33

How can snapshots of cotranslational folding at different points be taken?

Answer 33

By lengthening and shortening the gene artificially to change where it stalls and hence which bit of the nascent chain is protruding from the ribosomal exit tunnel.

Question 34

What is example of a protein whose N-terminal domains begin folding before the others have exited the ribosomal exit tunnel?

Answer 34

Ig2 antibodies. The first domain begins folding before even all of that has exited, and continues more after 37AAs of the linker region between the 106AA Ig domain is translated and the first domain is fully folded at a linker length of 89AA.

Question 35

How is NMR usually used to produce an image of the nascent protein?

Answer 35

By using N-15 tagged amino acids in the translation.

Question 36

How is the folding of ubiquitin thought to occur?

Answer 36

Beginning within the ribosomal exit tunnel.