RR12: Translation II

Question 1

How do tRNAs get to the A site of the ribosome?

Answer 1

They diffuse into the A site when the ribosome subunit is in its functional conformation and when the A site is free.

Question 2

What protein helps the charged tRNAs get to the A site?

Answer 2

tRNAs are associated with EF1alpha.
EF1alpha:
- elongation factor
- G protein (GTP-binding protein)

Question 3

What’s the role of EIF4?

Answer 3

It’s an elongation factor that binds to tRNAs and it plays a role in chain elongation during protein synthesis.

Question 4

What happens when a tRNA comes in the A site with its EF1A-GTP and the codon doesn’t correspond with the anticodon they have?

Answer 4

The tRNA will just diffuse out of the A site and try again later with another codon.

Question 5

What happens when the tRNA comes in the A site with its EF1A-GTP and the codon matches with its anticodon?

Answer 5

There’s a conformational change:
- GTP is hydrolyzed to GDP
- EF1A is released
- conformational change in the ribosome
- this change in the ribosome brings the amino acids linked to the tRNAs in the P and A sites into close proximity.

Question 6

What happens when the 2 amino acids attached to the tRNAs in site A and P come close?

Answer 6

They can interact.
A catalysis reaction happens:
- the alpha-amino group of the amino acid of the tRNA in the A site interacts with the terminal carboxyl group of the amino acid on the tRNA in the P site.
- Peptidyltransferase (enzymatic activity) forms a peptide bond between the alpha-amino group and the carboxyl group.

Question 7

Where does the peptidyltransferase activity happen?

Answer 7

It happens in the large subunit of the ribosome, in the region where we find the 23S rRNA (bacteria) or the 28S rRNA (eukaryote)

Question 8

What is the role of the 23S rRNA (bacteria) and 28S rRNA (eukaryotes)?

Answer 8

It catalyzes the peptide bond without any protein.

Question 9

Why can rRNA catalyze the peptide bond between the 2 amino acids?

Answer 9

Because the 23S (bacteria) et 28S (eukaryote) rRNA in the large subunit of the ribosome can fold up into secondary structures that can give rise to enzymatic activity by folding around their target.
They also have a ribozyme to mediate the reaction.

Question 10

What is a ribozyme?

Answer 10

It’s the only enzyme that’s not made up of proteins, it’s made up of rRNA.
It meditates the peptidyltransferase activity that creates a peptide bond between the 2 amino acids during the elongation of the aminoacid chain to form a protein.

Question 11

On which tRNA is the polypeptide chain linked to after the 2 amino acids are linked by a peptide bond?

Answer 11

It’s on the aminoacyl site, before the ribosome moves forward and then it’s on the peptide site.

Question 12

What happens after the peptide bond formation between the 2 amino acids?

Answer 12

There’s a conformational change in the ribosome and it moves forward one codon, 3 nucleotides.
The tRNA molecules move from A to P and from P to E.
The conformational change is due to the hydrolysis of EF1A from GTP to GDP when the anticodon matched the mRNA codon.
EF2 makes sure that the tRNAs translocate.

Question 13

Why can’t the complex go back on codon instead of going forward?

Answer 13

Because of hydrolysis. The GTP binding proteins make sure that the process goes forward.

Question 14

What happens after the translocation of the tRNAs?

Answer 14

EF2 gets hydrolyzed from GTP to GDP and the ribosome has a free A site for other tRNAs to diffuse and see if they match with the codon on the mRNA.

Question 15

Describe the elongation cycle of peptide chains.

Answer 15

1. 43S pre-initiation complex
2. eIF4 complex
3. Capping of mRNA by eIF4E
4. Binding of eIF4G to poly A tail to create an mRNA loop and to eIF3 on the preinitiation complex, linking the 2 complexes.
5. eIF4A makes the complex move to scan until it meets the AUG start codon
6. Hydrolysis of eIF2GTP to form the functional complex with the large subunit coming in and the other factors leaving.
7. tRNA (associated with EF1alpha-GTP) with complementary anticodon base-pairs with the A site
8. Hydrolysis of EF1alpha-GTP and release of EF1alpha leads to conformational change and the tRNA in P and A come close
9. Linkage of amino acid P and A by peptidyltransferase activity on the 23S or 28S rRNA in the large subunit with the help of ribozyme.
10. EF2-GTP makes the tRNAs translocate to the next site (1 codon)
11. Hydrolysis of EF2-GTP to make sure the complex doesn’t go back
12. A site is free to have another tRNA to keep making the chain grow.

Question 16

When the tRNAs get to the E site, do they still have an amino acid linked with them?

Answer 16

No. They don’t have anything and they’ll be pushed to leave when another tRNA comes to the A site.

Question 17

How fast is translation?

Answer 17

It happens at a rate of 3 to 5 amino acids per second.

Question 18

Is translation highly regulated?

Answer 18

Yes. You can’t make a mistake, because if EF2-GTP doesn’t do its job correctly and moves the tRNAs by 4 nucleotides, the codons won’t match the mRNA, so we won’t get the right protein.

Question 19

How does the elongation cycle of the polypeptide chain stop?

Answer 19

When the complex meets a stop codon.
There’s no tRNA made to interact with a stop codon: UAA, UAG, UGA.
The release factor (eRF1) gives rise to chain termination by recongizing the stop codon.

Question 20

WHat is eRF1?

Answer 20

Release factor.
It’s a protein that looks like a tRNA that can interact with the stop codon and make it stop.
It is associated with eRF3-GTP.
When eRF1 binds to the stop codon in the A site, eRF3- GTP gets hydrolyzed.

Question 21

What is the role of eRF3?

Answer 21

eRF3 in its GTP-bound form, is associated with eRF1. It gets hydrolyzed when eRF1 base-pairs with the stop codon in the A site.
Cleavage of the last amino acid in the P site from its tRNA.
It cleaves the polypeptide chain, so it can released, as well as the tRNA in the E site.

Question 22

What is the post-termination complex?

Answer 22

It’s the whole complex composed of the ADS ribosome, the mRNA, eRF1 associated with eRF3-GDP.

Question 23

When does the post-termination complex dissociate?

Answer 23

When the polypeptide chain is cleaved and when the tRNA in the E site is free to leave. The small subunit and the large subunit gets seperated.

Question 24

What happens as soon as the small and large ribosomal subunits dissociate after the elongation cycle of the polypeptide chain is done?

Answer 24

eIF3, eIF1 and eIF1A associate with the small subunit, so they can be ready to participate in another elongation cycle.

Question 25

Why does it matter that the mRNA is in a loop during translation?

Answer 25

Because the ribosome starts at the 5’ end where the eIF4 complex is to allow eIF4E to cap the 5’ end. Then, it translates the mRNA into a polypeptide chain to the 3’ end where the poly A tail is.
When it’s done, the ribosome dissociates and the large subunit, the small subunit and the initiation factors are all ready to start the translation of the same mRNA again.

Question 26

During translation, does it happen only one ribosome at a time?

Answer 26

No. Multiple ribosome can be on the same mRNA and make a lot of proteins. The more ribosomes the more proteins made.

Question 27

What’s a polysome?

Answer 27

It’s a cluster of ribosomes held together by a strand of mRNA that the ribosomes are all translating.

Question 28

How can we quantify the translational efficiency of an mRNA?

Answer 28

By knowing how many ribosomes are working to translate it into proteins.
We can know that by doing polysome profiles.

Question 29

How can we make polysome profiles?

Answer 29

We can use centrifugation to separate each of the ribosomal complexes working on one mRNA.
That way, we can see the lighter complexes at the top and the heavier complexes at the bottom.
We can separate:
- initiation factors
- the small ribosomal subunit
- the large ribosomal subunit
- the ADS ribosome
- polysomes, so depending on which one is at the bottom, we can figure out which mRNA has to most ribosomes, so which one is the most efficiently translated.