Initiation of protein synthesis in eukaryotes (Translation initiation in eukaryotes)
- Translation is generally similar in eukaryotic and bacterial cells. Most of the significant differences are seen in the mechanism of initiation.
- The initiation of translation in eukaryotes is complex as compared to prokaryotes.
- There are at least 12 well defined initiation factors involved in eukaryotic protein synthesis, and some have analogous functions to the three prokaryotic IFs.
- Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation.
- The prefix eIF denotes a eukaryotic initiation factor.
Energy requirement for translation initiation: Eukaryotic translation initiation consumes two ATPs (One for prokaryotic initiation).
- 1st ATP for unwinding the RNA duplex
- 2nd ATP for scanning along the mRNA to find start codon AUG
Major steps involved in the formation of 80S initiation complex
Step 1: Dissociation of 80S ribosomal subunits
Function of eIF6: Facilitate dissociation of inactive 80S ribosome into 40S and 60S subunit
The process begins with the binding of eIF3 and eIF1A (homolog of bacterial IF-1) to the 40S subunit.
Functions of eIF3: Binds to the 40S ribosomal subunit and inhibits its reassociation with the 60S subunit.
Function of eIF1A: Stimulate binding of eIF2 – GTP – Met tRNAiMet to 40S subunit.
Step 2: Formation of 43S pre initiation complex
43S pre initiation complex contains: Ternary complex + 40S subunit with bound eIF3 and eIF1A.
(Ternary complex: Complex containing three different molecules that are bound together).
Components of ternary complex: Met-tRNAiMet and elF-2 bound to GTP
Subscript “i”: Indicate initiator tRNA
- The presence of elF3 and elF1A stabilizes this 43S pre initiation complex.
- In the 43S complex the Met – tRNAiMet is not properly placed. This is because of the absence of mRNA.
Step 3: Recruitment of the 43S complex to the 5′ end of the mRNA by eIF3 and the eIF4 factors
The recruitment of 43S complex to the mRNA is mediated by the eIF4 group of factors.
eIF4F complex: Cap binding complex (heterotrimeric complex)
Components of Cap binding complex: eIF4E, eIF4G and eIF4A
Function of eIF4E: Binds to the 5’ Cap of mRNA (allows the 40S ribosomal subunit to bind to the 5′ end of an mRNA).
(The eIF4E is the cap-binding protein. It is the rate-limiting step of cap dependent initiation, and is often cleaved from the complex by some viral proteases to limit the cell’s ability to translate its own transcripts).
Function of eIF4G: Scaffold protein that mediates protein – protein interaction in the complex.
(Can interact with eIF4E, eIF4A, eIF3, and poly-A binding protein (Pab1))
It may be responsible for the synergistic effect of Cap and poly A-tail on translation.
The eIF4F–mRNA–eIF4B–eIF4H complex joins the 43S pre initiation complex through a protein–protein interaction between eIF4G and the 40S subunit-bound eIF3.
In eukaryotes pre-mRNA must be processed and transported to the cytoplasm before translation is initiated. Thus, there is a chance for the formation of complex secondary structures that must be removed to expose signals in the mature mRNA.
Function of eIF4A: ATP dependent RNA helicase that unwind RNA duplexes
Function of eIF4B: RNA binding protein that enhances the helicase activity of eIF4A
Function of eIF4H: RNA binding protein that enhances the helicase activity of eIF4A
For the appropriate association of 43S pre initiation complex with mRNA energy has to be supplied by ATP.
- The 3’ and 5’ ends of eukaryotic mRNAs are linked by a complex of proteins that includes several initiation factors and the poly A binding protein (PAB) thereby circularizing the mRNA.
Step 3: Scanning of the 5′ untranslated region (UTR) and recognition of the AUG codon
There is no Shine–Dalgarno sequence in eukaryotic mRNA. So the mechanism of selecting the start codon is different in eukaryotes.
Scanning hypothesis proposed by Kozak: The 40S subunit that already containing the initiator tRNA attaches to the 5’ – end of the mRNA and scans along the mRNA until it finds an appropriate AUG.
The consensus sequence for translation initiation is called Kozak sequence.
Kozak consensus sequence: 5’ GCCGCCACCAUGG 3’
A in the underlined AUG start codon is +1
For a ‘strong’ consensus, a purine in the –3 position and a G in the +4 position
The 43S pre initiation complex then translocates along the mRNA by an ATP-dependent process called scanning until it encounters the mRNA’s AUG initiation codon. This yields the 48S preinitiation complex.
The recognition of the AUG occurs mainly through base pairing with the CUA anticodon on the bound Met – tRNAi Met.
This explains why the initiator tRNA must bind to the small subunit before the mRNA (different in prokaryotes)
In almost all cases, eukaryotic mRNA has only one start site and hence is the template for a single protein.
- The initiating codon in eukaryotes is always AUG.
- Eukaryotes do not use a specific purine-rich sequence on the 5’ side to distinguish initiator AUGs from internal ones.
- Instead, the AUG nearest the 5’ end of mRNA is usually selected as the start site.
The start codon indicates the site where the mRNA will begin coding for the protein. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine.
Step 4: Assembly of the 80S ribosome (Recruitment of the 60S subunit to form the 80S initiation complex).
- The formation of the 48S preinitiation complex induces eIF2 to hydrolyze its bound GTP to GDP + Pi which results in the release of all the initiation factors.
- The hydrolysis reaction is stimulated by eIF5 which is acting as a GAP (GTPase activating protein).
Recycling of eIF2 – GTP complex to continue another round of initiation
Function of eIF2B: Recycling of the eIF2-GTP complex by exchanging GTP for GDP
Functions as eIF2’s GEF (guanine nucleotide exchange factor)
The process of initiation of translation in eukaryotes is of two types:
1) Cap-dependent initiation (The process described above)
2) Cap-independent initiation.
For details: Refer my lecture notes on cap – independent translation initiation