II. Post-transcription | 25A. Initiation of the translation in pro- and eukaryotes; regulation of eukaryotic translation; the role of phosphorylation of eIF2α

Question 1

1. Initiation of translation in prokaryotes

Answer 1

1. tRNA initiating the translation for prokaryotes becomes charged with N-formylmethionine (fMet-tRNA) – this will be the first amino acid
2. Small subunit (30S) binds to IF1 and IF3
3. Small subunit binds the mRNA together with IF2 (charged with GTP) and fMET-tRNA
   => Formation of 30S initiation complex, where the first added amino acid will be N-formylmethionine
4. IF1 & IF3 are released
5. Large subunit (50S) is brought in
6. IF2 hydrolyzes GTP, causing a conformational change = release of IF2
   => Formation of 70S initiation complex

Question 2

2. Initiation of translation in eukaryotes
   a/ What is the whole process of initiation of translation in eukaryotes?

Answer 2

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
1) Ribosomal dissociation
- Ribosomal dissociation from previous translation, subunits are kept apart by eIFs binding to small subunit (40S)

2) Formation of 43S pre-initiation complex
- Pre-initiation complex is formed when a 40S subunit with eIFs (1, 1A, 3) associates with eIF5 + ternary complex
- Ternary complex (tRNAiMet + eIF2 bound to GTP) will bind to the 40S subunit to form the 43S pre-initiation complex

3) mRNA-eIF4 complex
- The 43S pre-initiation complex will bind to the 5’ (methylguanosine) cap
- The mRNA binds to eIF4 complex with both 5’ cap and poly-A-tail
- This binding will result in the formation of a circular complex

4) Formation of 48S initiation complex
- The 43S pre-initiation complex slides along and scans the mRNA until the tRNAiMet anticodon recognize the start codon (AUG)
- Recognition of start codon -> hydrolysis to GTP (irreversible step) -> stops further scanning -> formation of 48S initiation complex

5) Formation of 80S initiation complex
- Binding of the large subunit (60S) to the complex (by eIF5) will lead to displacement of many other eIFs
- When all eIFs leave the complex, the large subunit (60S) and 48S initiation complex will join completely to form the 80S initiation complex
-> This is an irreversible step, since the ribosomal subunits do not dissociate until the entire mRNA is translated and protein synthesis over

Question 3

2. Initiation of translation in eukaryotes
   b/ Describe Ribosomal dissociation before initiation of translation in eukaryotes?

Answer 3

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
Ribosomal dissociation
- Ribosomal dissociation from previous translation, subunits are kept apart by eIFs binding to small subunit (40S)

Question 4

2. Initiation of translation in eukaryotes
   c/ Describe Formation of 43S pre-initiation complex

Answer 4

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
2) Formation of 43S pre-initiation complex
- Pre-initiation complex is formed when a 40S subunit with eIFs (1, 1A, 3) associates with eIF5 + ternary complex
- Ternary complex (tRNAiMet + eIF2 bound to GTP) will bind to the 40S subunit to form the 43S pre-initiation complex

Question 5

2. Initiation of translation in eukaryotes
   d/ Describe mRNA-eIF4 complex

Answer 5

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
3) mRNA-eIF4 complex
- The 43S pre-initiation complex will bind to the 5’ (methylguanosine) cap
- The mRNA binds to eIF4 complex with both 5’ cap and poly-A-tail
- This binding will result in the formation of a circular complex

Question 6

2. Initiation of translation in eukaryotes
   e/ Describe Formation of 48S initiation complex

Answer 6

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
4) Formation of 48S initiation complex
- The 43S pre-initiation complex slides along and scans the mRNA until the tRNAiMet anticodon recognize the start codon (AUG)
- Recognition of start codon -> hydrolysis to GTP (irreversible step) -> stops further scanning -> formation of 48S initiation complex

Question 7

2. Initiation of translation in eukaryotes
   f/ Describe Formation of 80S initiation complex

Answer 7

(eIF = eukaryotic initiation factor; tRNAiMet (initiator methionyl-tRNA))
5) Formation of 80S initiation complex
- Binding of the large subunit (60S) to the complex (by eIF5) will lead to displacement of many other eIFs
- When all eIFs leave the complex, the large subunit (60S) and 48S initiation complex will join completely to form the 80S initiation complex
-> This is an irreversible step, since the ribosomal subunits do not dissociate until the entire mRNA is translated and protein synthesis over

Question 8

Protein synthesis control point

Answer 8

Protein synthesis control point – phosphorylation of eIF2α:
- Ternary complex only forms when eIF2 associates with GTP, and tRNAiMet will only be bound when GTP associates with eIF2
- When the cell is under stress, phosphorylation of the α- subunit of the eIF inhibits translation initiation
- The phosphorylated complex cannot exchange the bound GDP for GTP – since eIF2 cannot bind tRNAiMet -> no protein synthesis will occur