Translation

Question 1

How long does mRNA usually last in a cell without particular protection from ribonucleoproteins?

Answer 1

It is usually only stable for several hours in physiological conditions, partly due to sequences within its code that promote degradation.

Question 2

How many tRNAs do pro and eukaryotes have?

Answer 2

Prokaryotes possess 30-40 different tRNA, eukaryotes have around 100.

Question 3

What is the structure of a tRNA?

Answer 3

74-95 bases long, these are ‘clover-shaped’ species with three arms: the anticodon loop, T-stem and aminoacyl acceptor stem,

The acceptor stem has a 3’ overhang called the 3’acceptor end which always ends with a CCA sequence, preceded by a single base called the discriminator base. This is involved in recognition specificity for the tRNA

Question 4

What loads tRNAs with AAs?

Answer 4

This is done by aminoacyl tRNA synthetases. There are 20 different version of this enzyme, each adding a different amino acid onto its appropriate tRNA to form an aminoacyl-tRNA. Each one must therefore be able to recognise all of the appropriate tRNAs for a given residue.

Question 5

What is the mechanism of AA loading?

Answer 5

To form the aminoacyl-tRNA, the enzyme adenylates the amino acid – forming a phosphoester bond at the C1 acid by removing two of the phosphate groups from ATP.

This is then displaced when the enzyme performs a transesterification, causing the AA to form a high energy ester bond with the alcohol on the end of the tRNA.

Question 6

What a=is the purpose of the aminoacyl tRNA synthetase editing pocket?

Answer 6

The editing pocket is used to allow for high accuracy and specificity, to ensure that the enzyme can discriminate between very similar residues (eg Valine & Isoleucine

Question 7

Wobble Base Pairing. What is that?

Answer 7

In prokaryotes there are fewer tRNA variants than there are codons. This means that some tRNAs must be able to recognise multiple codons (except for the ones for methionine and tryptophan, which have only one codon each). This is thought to be done using wobble base pairing, where the third base in the triplet codon can recognise multiple bases. This often utilises inosine.

Question 8

How fast are ribosomes?

Answer 8

Too fast.

Question 9

No, actually how fast are ribosomes?

Answer 9

Ugh.

Ribosomes can extend a polypeptide chain at a rate of 3-5 amino acids per second, meaning that they can produce a whole protein in a time frame that varies from minutes (100AA peptides - eg insulin, EGF) to hours (EGF receptor, titin).

Question 10

Is there like, a lot of ribosome hanging around or…?

Answer 10

The ribosome is the most abundant ribonucleoprotein in the cell, but the protein elements are purely for stability and structural integrity – all the catalysis is done by rRNA ribozymes.

So yeh, fuckloads.

Question 11

Where are ribosomes assembled?

Answer 11

the nucleolus

Question 12

What tells the ribosome where to begin translation?

Answer 12

In prokaryotes this is the Shine-Delgarno sequence (GGAGG) and in eukaryotes the Kozak sequence (CCACC).

Question 13

Where are the initiator AUGs in relation to the SD and kozak sequenci?

Answer 13

The Shine-Delgarno sequence is a 16S rRNA binding sequence found 5-9bp upstream of the AUG, whereas the Kozak sequence tends to be directly before the AUG.

And the correct plural is sequences. You jackass.

Question 14

Which residues do the different PTMs occur on?

Answer 14

• Phosphorylation – Ser, Thr, Tyr
• Glycosylation – Ser, Thr (O-Linked); Asn (N-linked)
• Methylation – Arg, Lys
• Acetylation – Lys
• Ubiquitination – Lys
• Hydroxylation – Lys, Pro

Question 15

What is the rate limiting step in translation/copulation?

Answer 15

Initiation

Question 16

By what two methods can ribosomes find the initiation sequence?

Answer 16

Cap dependent initiation in which the intiation complex itself scans the mRNA 5’-3’ until the start codon is found, or Internal Ribosome Entry Site (IRES) – in which the initiation complex binds upstream of the AUG codon in the middle of the 5’-UTR and the ribosome is responsible for the identification.

Question 17

Wait, what’s the initiation complex?

Answer 17

This is mostly comprised of eIFs (Eukaryotic Initiation Factors) that bind to the 7-methly guanosine cap on one end of the mRNA.

Question 18

How does the initation complex form?

Answer 18

The first protein to bind is eIF4E, which then recruits eIF4G. eIF4G binds to both eIF4A/B – which are responsible for binding to the ribosome – and PABP (PolyA Binding Protein) that circularises the mRNA.

This interaction ensures that the mRNA is complete before initiating translation, and increases the local concentration of important factors

Question 19

What is the ternary complex?

Answer 19

eIF1, 1A, 2, 3 and 5

Question 20

What is the 43S complex?

Answer 20

This is the 40S ribosome subunit complexed with a GTP-linked tRNA and the ternary complex.

Question 21

How is the 43S complex produced and what does it do?

Answer 21

eIF1A is responsible for generating a pool of 40S subunits, and with eIF3 binds the ternary complex to the 40S ribosome subunit producing the 43S complex. The 43S complex is responsible for scanning for the Kozak sequence.

Question 22

What is the final checkpoint before the 60S ribosome subunit is recruited?

Answer 22

Hydrolysis of the GTP bound to eIF2 by eIF5

Question 23

What happens after the 60S ribosome subunit is recruited?

Answer 23

The initiator methionine can then bind to the P site, which in prokaryotes is a special tRNA that has an aldehyde group attached to the amine. This is called N-formyl methionine (fMet).

Question 24

How does IRES work?

Answer 24

This remains unclear, but it is thought that the mRNA has a specificity region in the 5’-UTR that recognises a protein within the ternary complex called X.

Question 25

How can ribosomes bind other than using IRES or circularised and capped mRNA?

Answer 25

It is also for the recruitment to be dependent on the only the Cap or only the PolyA tail.

When only binding to the cap PABP is not required, and when only binding to the PolyA tail the Cap binding protein eIF4E is not needed.

Question 26

What is elongation?

Answer 26

This is the process by which new tRNAs are introduced into the A (aminoacyl) site, their residues peptide linked and then moved through to the P (peptidyl) site as the previous tRNA is detached in the E (exit) site and then released in the next cycle. The first Met-tRNA is already in the A-site from the initiation stage.

Question 27

What is the first step in elongation?

Answer 27

A̶r̶o̶u̶s̶a̶l̶?̶

This process begins with eEF2-GTP (eukaryotic elongation factor 2) entering the ribosome and using the hydrolysis of its GTP to push the Met-tRNAs through to the P site. eEF2-GDP then leaves the ribosome.

Question 28

What is the second step in elongation?

Answer 28

The new tRNAs enter the ribosome while bound to an eEIF1A-GTP factor. The eEIF1 hydrolyses the GTP to power the loading of the tRNA into the A-site, next to the existing tRNA in the P site. The eEIF1A-GDP is release, and its GDP is replaced with a fresh GTP by eEIF1B so that it can bind to a new aminoacyl-tRNA

Question 29

What is the final step in elongation?

Answer 29

The ribozyme peptidyl transferase moves the amino acid from the first tRNA in the P site to the new one in the A site and the process can begin again with eEIF2-GTP pushing the now empty tRNA out of the P-site and the tRNA holding the nascent amino acid chain into it, leaving the A-site open.

Question 30

What causes termination?

Answer 30

S̶c̶h̶w̶a̶r̶z̶e̶n̶e̶ nope.

This uses eRF1 (eukaryotic release factor 1) as a tRNA mimic, usually while bound to eEF1A

Question 31

How does eRF1 mimic tRNA?

Answer 31

The eRF1 possesses three domains (named 1, 2 and 3) that resemble the anticodon loop, aminoacyl acceptor stem and T-stem respectively

Question 32

How does eRF1 cause termination?

Answer 32

It binds to the A site at the stop codon (using domain 1), and then releases the protein chain by catalysing the hydrolysis of the ester bond between the P site tRNA and the peptide chain (using domain 3). It does this via nucleophilic attack stimulated by GTP bound eRF3

Question 33

How does termination differ between pro and eukaryotes?

Answer 33

In prokaryotes different RFs interact with the different stop codons, whereas in eukaryotes they are all recognised by eRF1.

Question 34

What RFs are present in prokaryotes, and what do they do?

Answer 34

• RF-1 – UAA and UAG
• RF-2 – UAA and UGA
• RF-3 – Similar role to eRF3, aiding ribosome binding using GTP

Question 35

How much energy is required for translation?

Answer 35

Energy is expended at all stages of translation, 1 ATP being used for initiation and 3 GTP being hydrolysed for each cycle.