Eukaryotes use a complex of many initiation factors

KEY TERMS:

• tRNA_i^Met is the special tRNA used to respond to initiation codons in eukaryotes.

KEY CONCEPTS:

• Initiation factors are required for all stages of initiation, including binding the initiator tRNA, 40S subunit attachment to mRNA, movement along the mRNA, and joining of the 60S subunit.
• Eukaryotic initiator tRNA is a Met-tRNA that is different from the Met-tRNA used in elongation, but the methionine is not formylated.
• eIF2 binds the initiator Met-tRNA_i and GTP, and the complex binds to the 40S subunit before it associates with mRNA.  

Initiation in eukaryotes has the same general features as in bacteria in using a specific initiation codon and initiator tRNA. Initiation in eukaryotic cytoplasm uses AUG as the initiator. The initiator tRNA is a distinct species, but its methionine does not become formylated. It is called tRNA_i^Met. So the difference between the initiating and elongating Met-tRNAs lies solely in the tRNA moiety, with Met-tRNA_i used for initiation and Met-tRNA_m used for elongation.

At least two features are unique to the initiator tRNA_i^Met in yeast; it has an unusual tertiary structure; and it is modified by phosphorylation of the 2 ribose position on base 64 (if this modification is prevented, the initiator can be used in elongation). So the principle of a distinction between initiator and elongator Met-tRNAs is maintained in eukaryotes, but its structural basis is different from that in bacteria (for comparison see Figure 6.13).

Eukaryotic cells have more initiation factors than bacteria—the current list includes 12 factors that are directly or indirectly required for initiation (for review see Dever, 2002). The factors are named similarly to those in bacteria, sometimes by analogy with the bacterial factors, and are given the prefix "e" to indicate their eukaryotic origin. They act at all stages of the process, including:

• forming an initiation complex with the 5 end of mRNA
• forming a complex with Met-tRNA_i
• binding the mRNA-factor complex to the Met-tRNA_i-factor complex
• enabling the ribosome to scan mRNA from the 5 end to the first AUG
• detecting binding of initiator tRNA to AUG at the start site
• mediating joining of the 60S subunit.

Figure 6.19 summarizes the stages of initiation, and shows which initiation factors are involved at each stage. eIF2 and eIF3 bind to the 40S ribosome subunit. eIF4A, eIF4B, eIF4F bind to the mRNA. eIF1 and eIF1A bind to the ribosome subunit-mRNA complex (for review see Pestova et al., 2001).

 

Figure 6.20 shows the group of factors that bind to the 5 end of mRNA. The factor eIF4F is a protein complex that contains three of the initiation factors (for review see Gingras, Raught, and Sonenberg, 1999). It is not clear whether it preassembles as a complex before binding to mRNA or whether the individual subunits are added individually to form the complex on mRNA. It includes the cap-binding subunit eIF4E, the helicase eIF4A, and the "scaffolding" subunit eIF4G. After eIF4E binds the cap, eIF4A unwinds any secondary structure that exists in the first 15 bases of the mRNA. Energy for the unwinding is provided by hydrolysis of ATP. Unwinding of structure farther along the mRNA is accomplished by eIF4A together with another factor, eIF4B. The main role of eIF4G is to link other components of the initiation complex.

eIF4E is a focus for regulation. Its activity is increased by phosphorylation, which is triggered by stimuli that increase protein synthesis, and reversed by stimuli that repress protein synthesis. eIF4F has a kinase activity that phosphorylates eIF4E. The availability of eIF4E is also controlled by proteins that bind to it (called 4E-BP1,2,3), to prevent it from functioning in initiation. eIF4G is also a target for degradation during picornavirus infection, as part of the destruction of the capacity to initiate at 5 cap structures (see 6.8 Small subunits scan for initiation sites on eukaryotic mRNA).

The presence of poly(A) on the 3 tail of an mRNA stimulates the formation of an initiation complex at the 5 end. The poly(A)-binding protein (Pab1p in yeast) is required for this effect. Pab1p binds to the eIF4G scaffolding protein(Tarun and Sachs, 1996). This implies that the mRNA will have a circular organization so long as eIFG is bound, with both the 5 and 3 ends held in this complex (see Figure 6.20) (for review see Sachs, Sarnow, and Hentze, 1997). The significance of the formation of this closed loop is not clear, although it could have several effects, such as:

• stimulating initiation of translation;
• promoting reinitiation of ribosomes, so that when they terminate at the 3 end, the released subunits are already in the vicinity of the 5 end;
• stabilizing the mRNA against degradation;
• allowing factors that bind to the 3 end to regulate the initiation of translation. 

eIF2 is the key factor in binding Met-tRNA_i. It is a typical monomeric GTP-binding protein that is active when bound to GTP, and inactive when bound to GDP (see 32.10 G proteins). Figure 6.21 shows that the eIF2-GTP binds to Met-tRNA_i (Asano et al., 2000). The product is sometimes called the ternary complex (after its three components, eIF2, GTP, Met-tRNA_i). 

Figure 6.22 shows that the ternary complex places Met-tRNA_i onto the 40S subunit. This generates the 43S initiation complex. The reaction is independent of the presence of mRNA. In fact, the Met-tRNA_i initiator must be present in order for the 40S subunit to bind to mRNA (for review see Hershey, 1991; Merrick, 1992). One of the factors in this complex is eIF3, which is required to maintain 40S subunits in their dissociated state. eIF3 is a very large factor, with 8-10 subunits. 

The next step is for the 43S complex to bind to the 5 end of the mRNA.Figure 6.23 shows that the interactions involved at this stage are not completely defined, but probably involve eIF4G and eIF3 as well as the mRNA and 40S subunit. eIF4G binds to eIF3. This provides the means by which the 40S ribosomal subunit binds to eIF4F, and thus is recruited to the complex. In effect, eIF4F functions to get eIF4G in place so that it can attract the small ribosomal subunit.

When the small subunit has bound mRNA, it migrates to (usually) the first AUG codon. This requires expenditure of energy in the form of ATP. It is assisted by the factors eIF1 and eIF1A. Figure 6.24 shows that the small subunit stops when it reaches the initiation site, forming a 48S complex.

Junction of the 60S subunits with the initiation complex cannot occur until eIF2 and eIF3 have been released from the initiation complex. This is mediated by eIF5, and causes eIF2 to hydrolyze its GTP. The reaction occurs on the small ribosome subunit, and requires the initiator tRNA to be base paired with the AUG initiation codon (Huang et al., 1997). Probably all of the remaining factors are released when the complete 80S ribosome is formed.

Finally the factor eIF5B enables the 60S subunit to join the complex, forming an intact ribosome that is ready to start elongation (Pestova et al., 2000). eIF5B has a similar sequence to the prokaryotic factor IF2, which has a similar role in hydrolyzing GTP (in addition to its role in binding the initiator tRNA).

Once the factors have been released, they can associate with the initiator tRNA and ribosomal subunits in another initiation cycle. Because eIF2 has hydrolyzed its GTP, the active form must be regenerated. This is accomplished by another factor, eIF2B, which displaces the GDP so that it can be replaced by GTP.

eIF2 is a target for regulation. Several regulatory kinases act on the α subunit of eIF2. Phosphorylation prevents eIF2B from regenerating the active form. This limits the action of eIF2B to one cycle of initiation, and thereby inhibits protein synthesis (for review see Dever, 2002).