TC - Translation (Initiation)

Question 1

What is Translation?

Answer 1

TRANSLATION is the process by which the genetic message, encoded in the sequence of the four RNA bases A, U, C and G is expressed in the form of an amino acid sequence in a protein using the 20 amino acids

Question 2

What do we need for protein synthesis? (6)

Answer 2

• A template that has clues on where to start decoding
• A supply of building blocks
• Something to assemble the building blocks into chains
• A way to supply the correct building block at the appropriate time
• Rules to define HOW to decode (including where to start and stop)
• Energy to drive the process

Question 3

General ribosome structure and the sites present (6)

Answer 3

1. Small and large subunits have to join otherwise no translation
2. Association creates three sites for tRNA to occupy
3. mRNA slides through a channel which is on small subunit

• A site (occupied by Aminoacyl-tRNA)
• P site (occupied by Peptidyl-tRNA)
• E site (occupied by deacylated tRNA, so Exit site)

Question 4

What are the main features of mRNA?

Answer 4

• Composed of ribonucleotides (so A, C, G and U)
• mRNAs contain non-coding or untranslated regions (NCRs, UTRs) at their 5’- and 3’-ends
• Template needs to define exactly where to start (determines ORF)

Question 5

Why is initiation crucial in translation?

Answer 5

It is the slowest step, limiting the rate of translation and is where most translational control occurs

Question 6

What occurs in Inititation with reference to initiation factors (IFs/eIFs)? (2)

Answer 6

1. Positioning of the small ribosomal subunit (and first aminoacyl-tRNA) at the initiation codon
2. Joining of large ribosomal subunit to make whole ribosome

Question 7

Protein synthesis in bacteria - Initiation

Answer 7

mRNA binds a special formylmethionine-tRNAf + the 30S subunit at the P-site using initiation factors IF1, IF2 and IF3 and GTP

• IF1 – binds in A site, prevents elongator tRNAs entering
• IF2 – binds the GTP and the fMet-tRNAf
• IF3 – prevents association with 50S, helps ensure fidelity of initiation codon selection (not present in all bacteria)

This gives the “initiation complex”

Question 8

How does the bacterial mRNA help the ribosome locate the initiation codon?

Answer 8

Prokaryotic mRNAs possess a Shine-Dalgarno sequence that base pairs to the 3’- end of the 16S rRNA

• This places the start codon AUG at the ribosome P-site about 10 bases 3’ of the S-D sequence

Question 9

What occurs after the ribosome locates the inititation codon? (3)

Answer 9

1. IF1 and IF3 dissociate once the 30S is at the initiation codon
2. This is accompanied by the binding of the 50S subunit and hydrolysis of the GTP bound to IF2, causing IF2 to also dissociate
3. This gives the 70S ribosome for elongation with the first tRNA in the P site, and an empty A site ready for the next tRNA

Question 10

Differences in eukaryotic messenger RNA (3)

Answer 10

• a cap (made from a modified G nucleotide) and a poly(A) tail
• No Shine-Dalgarno sequence
• 5’ and 3’ UTRs may contain several features (sequence or structure-based) that help regulate protein expression/mRNA stability/localisation

Question 11

What occurs in initiation in eukaryotes? (4)

Answer 11

1) eIF2 + the small 40S ribosome subunit + methionyl-tRNAimet + GTP bind to each other (43S pre-initiation complex)

2) The 40S subunit binds to mRNA with other initiation factors (eIFs) bound to the cap and poly A tail regions (48S pre-initiation complex)

3) The 40S ribosome then “scans” the mRNA looking for the AUG initiation codon:

• Usually uses first AUG it encounters

4) eIFs then dissociate and the 60S subunit binds

Question 12

What are some differences (2) and similarities (3) between eukaroytic and prokaryotic initiation?

Answer 12

Key differences:

• mRNA is circularised
• Complexity adds several opportunities for control

Similarities to prokaryotes:

• eIF2 bound to GTP and brings in first tRNA
• eIF3 and eIF1 ensure accuracy of initiation and prevent association of 60S large subunit

Question 13

What is the importance of correct protein folding? (2)

Answer 13

• Many proteins can fold spontaneously using only the primary amino acid sequence information
• Other proteins require assistance from chaperones

Question 14

What are Chaperones?

Answer 14

Heat Shock Proteins – prevent illicit liaisons between proteins (e.g. interactions between exposed hydrophobic regions)

Question 15

What happens to Malfolded proteins?

Answer 15

Malfolded proteins are (hopefully) rapidly degraded

• Protein misfolding in the ER can be a serious problem, and PERK exists to couple protein folding to protein synthesis

Question 16

Role of PERK in ER stress (3)

Answer 16

1. Normally, BiP binds to PERK and keeps it in an inactive monomeric state
2. Can elicit ER stress using brefeldin A (blocks protein processing) or thapsigargin (interferes with ER Ca2+)
3. BiP dissociates to bind to unfolded proteins, activating PERK dimers