Nucleic Acids 7- Translation and post-translational modification

Question 1

Why is Spatial and temporal coordination of transcription and translation important

Answer 1

Ensures that energy is now wasted on transcribing mRNA or translating proteins only for them to be degraded as they are not needed

Question 2

How is the DNA read

Answer 2

In a triplet code 300nt of exon= 100 amino acids

Question 3

What is a group of 3 nucleotides called

Answer 3

A codon

Question 4

Describe the typical structure of mRNA

Answer 4

5’ “cap” (7-Methyl Guanosine) – entry site for ribosome
UTR – untranslated regions – may be involved in regulation of translation or mRNA stability
polyA – protects mRNA from degradation
UTR present after 5’ cap util start codon
UTR also present after stop codon up until poly-A-tail

Question 5

What are the triplet codes that encode stop codons

Answer 5

UAA
UAG
UGA

Question 6

What does it mean if an amino acid has lots of codons that code for it

Answer 6

It is commonly used

Question 7

What is the start codon

Answer 7

Methionine (AUG)

Question 8

Describe simply how mRNA is read

Answer 8

Ribosome scans from 5’ end of mRNA (cap)
Translation starts at first AUG, continues in frame, i.e. with immediately succeeding triplet codon (CAU) and so on…..
Translation stops at first in frame termination codon

Question 9

Describe the structure of tRNA

Answer 9

RNA molecules can fold into 3-D structures by forming base pairs between different regions of the molecule, if these regions are extensive they will fold into double-helical molecules, this happens in tRNA, producing a molecules that looks like a cloverleaf. The cloverleaf undergoes further folding to form a L-shaped structure, further stabilised by Hydrogen bonds between the different regions.
There are two regions of unpaired nucleotides:
5’- anticodon
3’- amino acid.

Question 10

Describe the binding between the mRNA codon and the tRNA anticodon

Answer 10

Antiparallel binding, 5’-3’ 3’-5’

Complementary base pairing

Question 11

How does each tRNA molecule recognise the correct amino acid

Answer 11

Aminoacyl-tRNA synthetases covalently couple each amino acid to the appropriate set of tRNA molecules, different synthetase enzyme for each amino acid.
Aminoacyl-tRNA synthetases attach the amino acid to the 3’ end of the amino acid, coupled by the hydrolysis of ATP– AMP +PPi. This process is known as charging and it produced a high energy bond between the charged tRNA and the amino acid, the energy of this bond can be used later to form peptide bonds between adjacent amino acids.

Question 12

What are the steps of translation

Answer 12

Initiation, elongation, termination

Question 13

What is the function of ribosomes

Answer 13

Hold the tRNAs in position
Capture complementary tRNAs
Move along the mRNA

Question 14

What is the structure of a ribosome

Answer 14

In eukaryotes- 80s. Made up of one 40S unit and one 60S unit

Question 15

Describe simply the steps of initiation

Answer 15

Step 1: dissociation of ribosome subunits (eukaryotic 40S + 60S)

Step 2: assembly of preinitiation complex
containing Met-tRNA + eIFs + 40S subunit

Step 3: binding of mRNA to preinitiation complex

Step 4: binding of 60S subunit

Question 16

Describe the assembly of the pre-initiation complex

Answer 16

eIF4E and eIF4G bind to cap and are recognised by 40S/Met-tRNA/eIF2
Only initiator Met-tRNA can bind to 40S subunit alone
40S subunit is primarily involved in tRNA and mRNA recognition- searches for AUG start codon of mRNA.

Question 17

Describe the binding of the mRNA to the pre-initiation complex

Answer 17

initiator Met binding sets the frame of the translation

Question 18

Describe the binding of the 60 S subunit

Answer 18

Translation initiation factors dissociate

GTP— GDP + Pi (ensures correct pairing)

Question 19

Where is the Met-tRNA found

Answer 19

On the P site of the 40S subunit.

Question 20

What happens to methionine when the final polypeptide is synthesised

Answer 20

It is usually removed by a specific protease.

Question 21

Describe, simply, the process of elongation

Answer 21

Step 1: binding of new tRNA carrying second amino acid to “amino acyl” (A) site

Step 2: catalysis of peptide bond formation between the two amino acids by peptidyl transferase (PT) on the large ribosomal subunit

Step 3: translocation of tRNA to P site and dissociation of first tRNA

Question 22

How is the charged tRNA carrying the next amino acid added.

Answer 22

It binds to the vacant A site of the ribosome, where it base pairs with the mRNA codon that is based there
The next mRNA codon is in frame as the A and P sites are sufficiently close that they form base pairs with mRNA codons that are contiguous, meaning that there are no stray bases between them.

Question 23

Describe the formation of the peptide bond between adjacent amino acids

Answer 23

The carboxyl group of the amino acid is uncoupled from the tRNA at the P site and is joined by a polypeptide bond to the free amino group of the amino acid bound to the tRNA at the A site.
This reaction is catalysed by peptidyl transferase (PT) on 60S subunit

Question 24

Describe the translocation of the large subunit

Answer 24

The shift moves the two tRNAs to the E and P sites respectively.
Elongation Factors (EFs) are proteins that promote movement of ribosome along mRNA using GTP

Question 25

Describe the translocation of the small subunit.

Answer 25

The shift moves the small subunit back to its original position relative to the large subunit, and it moves exactly 3 nucleotides along the mRNA molecule, hence ejecting the spent tRNA that was previously on the E site, this resets the ribosome, vacating the A site.

Question 26

Describe the importance of GTP hydrolysis in elongation.

Answer 26

EFs use the energy of GTP to enhance the efficiency and accuracy of translation by providing “pauses” (e.g. GTP hydrolysis) that allow incorrect base pairs to dissociate

Question 27

Describe, simply, the process of termination

Answer 27

Step 1: recognition of stop codon

Step 2: release of peptide chain

Step 3: dissociation of release factors and ribosomes

Question 28

Describe how a stop codon is recognised

Answer 28

Release factors (proteins, not tRNAs) bind to empty A site – there is no tRNA for stop codons
This activates peptidyl transferase which adds a water molecule to the peptidyl tRNA instead of an amino acid

Question 29

Describe how the polypeptide chain is released

Answer 29

This reaction frees the carboxyl end of the final amino acid from its attachment to tRNA, hence releasing the completed protein chain, as this was its final attachment to the ribosome.

Question 30

What happens after the polypeptide chain is released

Answer 30

60S and 40S units dissociate. The release factors dissociate.
Available for another round of translation.

Question 31

Describe the concept of polyribosomes

Answer 31

Ribosomes do not work singly on a mRNA but in multiple copies on the mRNA – a polyribosome – like a string of beads.
Multiple ribosomes synthesising simultaneously a 300 amino acid long protein, allowing many copies of the protein to be produced.

Question 32

Describe how antibiotics disrupt protein synthesis

Answer 32

Translational machinery is complex, easily disrupted – common target for antibiotics
• Antibiotics exploit differences between prokaryotic and eukaryotic ribosomes and translation factors
Antibiotics selectively inhibit prokaryotes
Antibiotics are natural products of bacteria or fungi to give them a selective advantage over other microbes

Question 33

Give some examples of antibiotics that inhibit protein synthesis

Answer 33

Streptomycin Inhibits initiation
Tetracycline Inhibits aa-tRNA binding
Erythromycin Inhibits translocation
Chloramphenicol Inhibits peptidyl transferase
Puromycin Terminates elongation prematurely

Question 34

Describe post-translational modifications

Answer 34

• After synthesis, most proteins are modified further before they are fully functional- Only 20 amino acids – cell uses post-translational modifications (over 200) to increase diversity, including:
  Disulphide bond formation (e.g. insulin)
  Proteolytic cleavage (e.g. insulin -> A and B chains)
  Addition of carbohydrate (Glycosylation)
  Addition of phosphate (Phosphorylation)
  Addition of lipid groups (Prenylation, Acylation)
  Hydroxylation (e.g. Collagen; Leitinger lecture)

Question 35

Describe the post-translational modification of insulin

Answer 35

ER-preproinsulin
ER/Golgi- Signal sequence cleaved- proinsulin- disulphide bonds form between chain B and chain A and within chain A
SV- C-chained removed- Insulin

Question 36

Describe the importance and role of the signal sequence

Answer 36

First 20-24 amino acids = “signal sequence”
(enriched in hydrophobic amino acids, e.g. Leu, Ile, Phe, Trp, Tyr, Ala)
Step 1: recognition of signal sequence by a protein-RNA complex “Signal Recognition Particle” (SRP), halting translation
Step 2: binding of SRP to a receptor at the RER surface, translation resumes
Step 3: translocation of the growing polypeptide into the lumen of RER
Step 4: cleavage of signal sequence by signal peptidase (co-translational) and folding- degraded in cytoplasm

Question 37

What are the features of transmembrane proteins

Answer 37

Transmembrane proteins encode one or more extra hydrophobic sequences that hold them in the membrane