Translational Control

Question 1

Where are proteins synthesised in eukaryotes?

Answer 1

polyribosomes

Question 2

What does each ribosome subunit do?

Answer 2

• large (60S) - catalyses peptide bond formation
• small (40S) - decodes the genetic message

Question 3

What are components of a polysome?

Answer 3

• mRNA template
• ribosomes
• tRNAs
• various protein factors
• nascent polypeptides

Question 4

Describe the ribosome scanning model

Answer 4

small ribosome subunit scans for first AUG codon in favourable context (Kozak consensus)

Question 5

Where does translational initiation occur from?

Answer 5

the first AUG codon

Question 6

What are the 5 steps of translational initiation?

Answer 6

1.tRNA binds to elF2 and small ribosome subunit (P site)
2. binds to the mRNA cap preoccupied with initiation factors elF4E and elF4G
3. scans for the first AUG
4. elF2 dissociates and large ribosome subunit binds
5. second aminoacyl-tRNA is added with a correct anticodon

Question 7

What does each ribosome have?

Answer 7

3 binding sites for tRNAs and 1 binding site for mRNA

Question 8

What 4 components does a tRNA have?

Answer 8

• acceptor stem - amino acid attachment site
• D arm/loop - recognition site for aminoacyl-tRNA synthetase
• T arm/loop – recognition site for ribosome
• anticodon – pairing with codon

Question 9

What is a codon?

Answer 9

a sequence of 3 consecutive nucleotides in a DNA or RNA molecule that codes for a specific amino acid

Question 10

What do codon wobbles positions allow?

Answer 10

one tRNA to recognise multiple codons

Question 11

What are the 5 steps of elongation?

Answer 11

1. aminoacyl-tRNA molecule binds to a vacant site A on the ribosome
2. peptide bond formation
3. large subunit translocates, leaving the 2 tRNAs in hybrid sites
4. small subunit translocates, carrying its mRNA 3 nucleotides through the ribosome
5. this resets the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA to bind

Question 12

How is each amino acid added to the growing polypeptide chain selected?

Answer 12

by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain

Question 13

What direction is mRNA translated?

Answer 13

5’ → 3’ with the N-terminal end of a protein being made first

Question 14

What do translation factors do?

Answer 14

drive translation in the forward direction

Question 15

What does Ef-Tu do?

Answer 15

provide opportunities for proofreading of the codon–anticodon match which allows incorrectly paired 5′ tRNAs to be selectively rejected, and the accuracy of translation is improved

Question 16

What does binding of EF-G to the ribosome and GTP hydrolysis cause?

Answer 16

a conformational change of the ribosome which moves tRNA to the P-site after formation of the peptide bond

Question 17

What are the 3 steps of proofreading by 16s rRNA?

Answer 17

1. 16S rRNA recognises correctly paired anticodon-codon
2. triggers GTP hydrolysis by EF-Tu
3. EF-Tu is released to proceed for protein synthesis

Question 18

How is translation terminated?

Answer 18

by the binding of release factor to an A site bearing a stop codon

Question 19

What are the 3 eukaryotic termination codons?

Answer 19

UAA, UAG and UGA

Question 20

What are the 4 steps in the creation of functional proteins?

Answer 20

1. protein folding
2. binding of small molecule cofactors for proper folding
3. covalent modifications
4. formation of complex

Question 21

How are proteins folded into a stable 3D structure?

Answer 21

by burying most hydrophobic residues into an interior core through a large number of non-covalent interactions between various parts of the protein

Question 22

What happens to incorrectly folded polypeptides?

Answer 22

they are destroyed by proteasomes

Question 23

What do many newly released proteins have?

Answer 23

molten globules which can be further folded

Question 24

What does the protein folding pathway use?

Answer 24

the molecular chaperones Hsp70 and Hsp60

Question 25

What do Hsp70 and Hsp60 do?

Answer 25

- work with a small set of associated proteins - bind to exposed hydrophobic patches on incompletely folded proteins - hydrolyse ATP

Question 26

When do Hsp70 and Hsp60 act respectively?

Answer 26

- Hsp70 - before translation is completed - Hsp60 - after completion of protein synthesis

Question 27

What are the 4 steps of the Hsp70 protein folding pathway?

Answer 27

1. Hsp70 binds to a string of 4-5 hydrophobic amino acids 2. it hydrolyses ATP to ADP and clamps down tightly on target 3. rebinds ATP and releases target protein 4. incorrectly folded protein may be tried again by the Hsp70 machinery

Question 28

What are the 7 steps of the Hsp60 protein folding pathway?

Answer 28

1. Hsp60 forms large barrel-shaped structure 2. captures a misfolded protein by hydrophobic interaction with the exposed surface 3. ATP binding 4. addition of a cap protein (GroES) to increase the dimension of the barrel rim 5. incubation for ~10s 6. ATP hydrolysis to weaken cap binding 7. additional ATP binding to eject folded protein

Question 29

What is the proteasome made up of?

Answer 29

- 20S core – multiple subunits that form a cylindrical stack of 4 heptameric rings and active sites on the interior surface - 2 19S caps – complex of ~20 subunits that recognises ubiquitinated and unfolded proteins

Question 30

Where is the proteasome present?

Answer 30

throughout the cytosol and nucleus to attack ubiquitin labelled proteins

Question 31

What is the proteasome?

Answer 31

a compartmentalised protease with sequestered active sites

Question 32

What are the 3 steps of proteasome action?

Answer 32

1. improperly folded proteins are targeted by attachment of a stretch of ubiquitin 2. ubiquitin-tagged proteins are translocated to proteosomes, and ubiquitin is removed from the cap by ubiquitin hydrolase for recycling 3. targeted proteins are unfolded in the ring at the cap and threaded into the core (cylinder) for degradation

Question 33

What is the difference between a proteasome and a common protease?

Answer 33

protease cuts once for each binding and does not require ATP whereas the proteasome cuts the entire protein substrate multiple times into short peptides

Question 34

What do many unstable mRNAs have?

Answer 34

AU rich sequence in their 3’ UTRs

Question 35

What prevents translation from taking place?

Answer 35

deadenylase shortens the polyA tail

Question 36

What do actively translated mRNAs tend to have?

Answer 36

longer half-lives

Question 37

What does codon composition affect?

Answer 37

polyA tail length, translation efficiency and mRNA stability

Question 38

What do mRNAs enriched in optimal codons have compared to genes enriched in non-optimal codons?

Answer 38

longer polyA tails, higher translation efficiency and higher stability

Question 39

What are the 2 codes interpreted by the ribosome within the mRNA?

Answer 39

- genetic code which specifies the amino acid sequence - conserved codon optimality code that shapes mRNA stability and translation

Question 40

Give examples of specialised mRNA decay mechanisms

Answer 40

- iron starvation - excess iron

Question 41

What are miRNAs?

Answer 41

small RNAs transcribed by RNAPII with a cap and polyA tail

Question 42

What do miRNAs do?

Answer 42

target specific mRNA by base pairing which leads to degradation of mRNAs or translation block

Question 43

What are the 4 steps of mRNA degradation by miRNAs?

Answer 43

1. miRNAs transcribed by RNAPII and exported to cytoplasm 2. dicer (RNase) cleaves miRNA to retain the targeting miRNA 3. targeting miRNA forms RISC with Argonaute and other proteins 4. RISC targets specific mRNAs based on base-pairing