7. Eukaryotes use a complex of many initiation factors

6.7 Eukaryotes use a complex of many initiation factors

The process of initiation in eukaryotes appears to be generally analogous to that in E. coli. Initiation in eukaryotic cytoplasm almost always 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.

Figure 6.13 fMet-tRNA_f has unique features that distinguish it as the initiator tRNA.

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 Xat least nine already have been found in reticulocytes (immature red blood cells), in which the most work has been done. 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.

Figure 6.18 In eukaryotic initiation, eIF-2 forms a ternary complex with Met-tRNA_f. The ternary complex binds to free 40S subunits, which attach to the 5 F end of mRNA. Later in the reaction, GTP is hydrolyzed when eIF-2 is released in the form of eIF2-GDP. eIF-2B regenerates the active form.

Eukaryotic initiation proceeds through the formation of a ternary complex containing Met-tRNA_i, eIF2, and GTP. The complex is formed in two stages. GTP binds to eIF2; and this increases the factor’s affinity for Met-tRNA_i, which then is bound. Figure 6.18 shows that the ternary complex associates directly with free 40S subunits. 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 435, 438).

Figure 6.19 Several eukaryotic initiation factors are required to unwind mRNA, bind the subunit initiation complex, and support joining with the large subunit.

The roles of the initiation factors involved in binding the subunit initiation complex to mRNA are expanded in Figure 6.19.

When the small subunit has bound mRNA, it migrates to (usually) the first AUG codon. Little is known about this process; we assume it requires expenditure of energy in the form of ATP. When the small subunit reaches the initiation site, it stops, and can be joined by a large subunit.

The factor eIF4F is a protein complex of many subunits that regulates the key events in recruiting ribosomes to mRNA (for review see 994). It binds at the 5′ end of the mRNA. 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 RNA-dependent ATPase 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 sythesis. eIF4F contains a subunit (called Mnk1) which is a kinase that acts to phosphorylate eIF4E. The availability of eIF4E is also controlled by proteins that bind to it (called 4EBP1,2,3), to prevent it from functioning in initiation.

How does the ribosome recognize the mRNA? eIF4G binds to eIF3, which is associated with the small ribosomal subunit. 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. Other factors (including eIF4A and eIF4B) are also involved.

Factors eIF3 and eIF6 are required to maintain subunits in their dissociated state. eIF3 is a very large factor, with 8 V10 subunits. eIF3 binds to the small subunit, but eIF6 (which has most of the antiassociation activity) binds to the large ribosomal subunit. It is released when the large subunit joins the initiation 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, which is a GTPase. Probably all of the remaining factors are released when the complete 80S ribosome is formed

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 eIF4G, which in turn is bound to eIF4E (see Figure 6.19). This implies that the mRNA must (transiently) have a circular organization, with both the 5′ and 3′ ends held in this complex (for review see 439).

Reviews
435:	Hershey, J. W. B. (1991). Translational control in mammalian cells. Ann. Rev. Biochem 60, 717-755.
438:	Merrick, W. C. (1992). Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291-315.
439:	Sachs, A., Sarnow, P., and Hentze, M. W. (1997). Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 89, 831-838.
994:	Gingras, A. C. , Raught, B. , and Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.. Ann. Rev. Biochem 68, 913-963.