Figure 15.11. Nucleotide sequence and secondary structure of the eukaryotic initiator tRNA (left) (M. Simsek & U. L. RajBhandary, Biochem. Biophys. Res. Commun. 49: 508-515, 1972), compared with the eukaryotic methionine tRNA participating in elongation (right) (H. Gruhl & H. Feldman, FEBSLetters 57: 145-148, 1975). Note a unique 2'-phosphoribosyl adenosine in the T stem (position 64), a "triple A" (positions 20, 54 and 60 clustered together in tertiary structure), and three consecutive G:C pairs in the anticodon stem of the initiator tRNA.

which feature of the animal initiator tRNA structure (where position 64 is not modified) takes the part of the negative discriminant for eEF1A.

In contrast to the initiator tRNA of Prokaryotes and mitochondria, the 5'-terminal nucleoside residue of the eukaryotic cytoplasmic initiator tRNA is paired with the nucleoside residue of the 3'-terminal region, just as in all elongator tRNAs. However, it is always the "weak" A1:U72 pair in the initiator tRNAs of animals, plants and fungi. Such a pair at the end of a helix can be easily disrupted, like in the case of the interaction of aminoacyl-tRNAs with the class I aminoacyl-tRNA synthetases (see Section 3.3). An attractive model is that it is disrupted at the interaction with eIF2, and then the 3'-tail of the Met-tRNAi bends back and thereby enables the terminal adenosylmethionine residue to lie on the helical acceptor stem (Basavappa & Sigler, 1991). In such a case the methionine side chain could positively contribute to the recognition of Met-tRNAi by eIF2.

15.3.5.Ribosomal Initiation Factors

The eukaryotic initiation factors (for reviews, see Merrick, 1992; Merrick & Hershey, 1996) can be conditionally divided into two principal groups: those which bind and operate with ribosomal particles promoting ribosomal subunit dissociation/association, initiator Met-tRNAi binding and mRNA binding, and those which are aimed at mRNA and engaged in preparing its upstream region for initiation. The first group (which is called here ribosomal initiation factors) contains the factors analogous to prokaryotic IF1,

IF2 and IF3, namely eIF1 (and eIF1A), eIF2 and eIF3, respectively, as well as several additional factors, such as eIF2B and eIF5. The second group which is to be considered in the next Section, seems to have no analogs in Prokaryotes and includes special mRNA-binding and mRNA-unwinding proteins facilitating initiation of translation; these are, first of all, the factors of eIF4 group, namely eIF4A, eIF4B, eIF4F and eIF4E. Mammalian initiation factors are considered below.

eIF1 is a small protein of a molecular mass of 12.6 kDa. It seems to be an auxiliary factor, like the prokaryotic IF1, stimulating formation of and stabilizing the initiation complexes of the small (40 S) ribosomal subunit. There is another initiation factor, designated now as eIF1A and earlier called eIF-4C, also taking part in the assembly of the initiation complexes of the 40S ribosomal subunit; it is a small acidic protein of molecular mass of 16.5 kDa.

eIF2, in contrast to its prokaryotic functional analog, is a complex protein consisting in three different subunits: acidic, 36.1 kDa (a); acidic, 38.4 kDa (P); and basic, 51.8 kDa (g). Like the prokaryotic IF2, it interacts with GTP and initiator Met-tRNAi. The main function of eIF2 is GTP-dependent binding of the initiator Met-tRNAi to the initiating 40S ribosomal subunit (for reviews, see Voorma, 1991; Trachsel, 1996). eIF2 is also a target of the regulatory phosphorylation by specific kinases which results in the inhibition of the rate of initiation complex formation (Section; the phosphorylation site is Ser51 on the a-subunit. The p-subunit seems to bear the mRNA-binding domain. The g-subunit is strongly homologous to EF1A, especially in its G-domain, and may be mainly responsible for both GTP binding and Met-tRNA binding. At the same time, GTP analogs and Met-tRNA can cross-link to the p-subunit as well, this suggesting its close contact with the g-subunit and its direct or indirect participation in the binding of the ligands.

Apart from its function to bind GTP and initiator Met-tRNAi, eIF2 has been reported to be capable of recognizing a specific initiation site in some mRNAs. This mRNA-binding activity resides in the a-subunit of eIF2 and is regulated by ATP which interacts also with the p-subunit and switches on its mRNA binding activity. No ATP hydrolysis takes place in this case. It has been demonstrated that the binding of eIF2 to Met-tRNAi with GTP and the binding to mRNA with ATP are mutually exclusive, although distinct epitopes of eIF2 are involved in the two binding activities. It can be speculated that, once bound to the 40S ribosomal subunit, eIF2 with ATP may interact directly with mRNA and thus guide the 40S subunit to its binding site in mRNA, and then GTP switches the activity of eIF2 on Met-tRNAi binding.

eIF2A and eIF2B are additional proteins promoting the functions of eIF2 and having no analogs in Prokaryotes. eIF2A is a simple basic protein of a molecular mass of 65 kDa. It may take part in AUG-dependent Met-tRNAi binding to the initiating 40 S ribosomal subunit. eIF2B is a large multi-subunit protein consisting in 5 different subunits with molecular masses of 33.7 kDa (a), 39 kDa (p), 58 kDa (g), 57.1 kDa (8) and 80.2 kDa (e). It is capable of forming a complex with eIF2 outside the ribosome and facilitates GDP/GTP exchange on eIF2 (see below, Section 15.3.8, Fig. 15.15); the regulation of the availability of the active (GTP-bound) form of eIF2 in the eukaryotic cell may be a possible function of eIF2B.

eIF3 is the largest initiation factor in Eukaryotes. It is a complex multi-subunit protein with a total molecular mass of about 600 kDa. At least 8 different subunits compose the protein; the molecular masses of the subunits are 35 kDa (a), 36.5 kDa (p), 39.9 kDa (g), 46.4 kDa (8), 47 kDa (e), 66 kDa (£), 105.3 kDa (h) and 170 kDa. (8). The protein has a strong affinity for the 40S ribosomal subunit, promotes dissociation of terminated ribosomes into subunits and forms the "native" 43S particles (complexes with 40S ribosomal subunits) competent to begin the initiation process. Thus eIF3 is an initiation factor which is necessarily present on the 43S initiation complex. This multi-subunit factor is also known as a strong mRNA-binding protein. However, the interaction of eIF3 with mRNAs and other polyribonucleotides has been shown to be non-specific. Nevertheless, the non-specific RNA-binding capacity of eIF3 may be exactly what is needed for the interaction of the 43 S initiation complex with mRNA, provided the specificity is introduced by other proteins.

A protein of 25 kDa called eIF3A or eIF6 has been reported to form a complex with dissociated 60S ribosomal subunit ("native" 60S particle).

eIF5 is an initiation factor having no analogs in Prokaryotes. Two, seemingly unrelated, forms of eIF5 have been described: p150 and p45. Both forms of eIF5 have been reported to induce GTPase on the 40S-bound eIF2 and the release of eIF2 (and possibly other initiation factors) in the process of 60S ribosomal subunit joining to the 43S initiation complex. In Prokaryotes these functions are fulfilled by the large ribosomal subunit itself. One more initiation factor, eIF5A, earlier called eIF4D, which is a small acidic protein of molecular mass 15 kDa, may also contribute to the ribosomal subunit joining and correct 80S ribosome formation.

15.3.6.mRNA-binding Initiation Factors

Factually the mRNA-binding initiation factors of the eIF4 group may form a complex functioning as a whole. Indeed, eIF4E is an essential subunit of eIF4F. eIF4A is another subunit of eIF4F, but bound loosely and also existing in excess as free protein. eIF4B is not considered as a part of eIF4F, but they have a significant affinity to each other and function rather in association. Three main successive functions of the eIF4 complex are (1) recognition of the cap structure of mRNA, (2) unwinding of cap-adjacent sequence of mRNA, and (3) facilitating the proper landing of initiating 40S ribosomal subunit (the so-called 43S initiation complex) on mRNA (reviewed by Rhoads, 1991). Correspondingly, three activities of the eIF4 complex should be mentioned: ATP-independent cap-binding activity, ATP-dependent RNA helicase activity, and an affinity to the ribosomal 43S initiation complex. It should be added that mRNA-binding capacity is clearly manifested also by eIF2 and eIF3. Being mostly attached to the "native" 40S ribosomal subunits, they may also take part in the proper landing of the ribosomal initiation complex on mRNA. Complex

Initiation factor eIF4F specifically binds to the cap structure of eukaryotic mRNAs. Three subunits are usually accepted to be components of the mammalian factor: a (p25, or eIF4E) is the subunit responsible for cap recognition, b (p45) is RNA-dependent ATPase and identical to free eIF4A, and g (p220, or eIF4G; real molecular mass is of 153.4 kDa in mammals) is a core protein seemingly also participating in initial mRNA binding. The b subunit (eIF4A) is loosely bound in the complex and can be lost during isolation and purification procedures. No eIF4A was found in eIF4F preparations isolated from some sources, such as wheat germs. In any case, eIF4A subunit can be omitted from eIF4F without loss of the cap-binding activity, so that the association of eIF4G (p220) with eIF4E can be considered as a minimal cap-binding complex. eIF4E is bound with the N-terminal portion of eIF4G, whereas eIF4A (and eIF3, see below) may associate with the C-terminal part of this large subunit.

It is interesting that in wheat germ cells two different cap-binding complexes have been found. One is similar to the mammalian eIF4F having p26 (eIF4E) and p220 (eIF4G) subunits, whereas the other consists of antigenically unrelated subunits p28 and p80. The "typical" eIF4F is represented in a considerably less amount in wheat germ cells as compared with the "iso"-eIF4F.

eIF4F is an RNA-binding protein in the sense that it has some affinity to any RNA, but it strongly binds specifically to the cap structure of eukaryotic mRNAs due to the presence of the cap-recognizing p25 subunit (eIF4E). This binding is ATP-independent. eIF4E seems to be the most deficient polypeptide among initiation factors and their subunits in eukaryotic cells, so that the cap-binding complex as a whole (eIF4F) is present in a limiting amount. In this situation cytoplasmic mRNAs must compete for eIF4F binding. RNA Helicase Complex

Immediately after the cap-binding step, or already during the binding, the unwinding of the cap-adjacent region of mRNA starts. This step is ATP-dependent. Thus, the RNA helicase reaction takes place. There are two helicase complexes: the combination of eIF4F(abg) with eIF4B, and the combination of free eIF4A with eIF4B. In both cases two polypeptides seem to be directly engaged and critical for the process: eIF4A and eIF4B.

Similarly, the RNA helicase reaction occurs in the process of cap-independent, internal initiation. After the recognition of the internal ribosome entry site (IRES) on mRNA by the ribosomal initiation complex, the same helicase complexes may go into action and unwind the downstream mRNA region for scanning and initiation. Specifically, eIF4F binds to IRES in a cap-independent manner, probably due to the affinity of the central part of eIF4G to IRES structure (Pestova et al., 1996).

Both ATPase and unwinding activities seemingly reside in eIF4A. eIF4A is a 45 kDa protein which is the most abundant initiation factor in eukaryotic cells. It exists in a free state and also as a labilely associated subunit of eIF4F. In eIF4F it is bound with the C-terminal portion of eIF4G (p220, or g-subunit of eIF4F). eIF4A, being alone or in complex with eIF4G, is capable of binding to single-stranded regions of RNA in an ATP-dependent manner and exhibits an RNA-dependent ATPase activity, but eIF4B strongly stimulates both effects. The continuously repeating cycles consisting in the ATP-dependent binding to a single-stranded mRNA site, the mRNA-induced ATP cleavage and the resultant eIF4A release (Fig. 15.12) may dynamically maintain the single-stranded state of a proper region of mRNA, preventing its refolding and its complexing with competing RNA-binding proteins.

The amino acid sequence of eIF4A demonstrates that it belongs to the so-called DEAD family of proteins which are present in various compartments of the eukaryotic cell and involved in different cellular processes. All are believed to possess ATPase and RNA unwinding activities. There are several conserved amino acid sequence regions in common, including two motifs (AXXXXGKT and DEAD) very characteristic of ATPases. Thus, eIF4A can be considered as the catalytic ATPase/unwinding subunit of the helicase initiation complexes.

eIF4B is a larger protein, of about 80 kDa, rather limited in its amount in the cell. It can be associated with eIF4F but in an even looser way than eIF4A. According to its primary structure, it bears a typical RNA-binding domain (the so-called "RNP consensus site", or RNP-CS), with two conserved sequence motifs (the so-called "RNP-1" and "RNP-2" motifs), characteristic also of poly(A)-binding protein, hnRNP A1 and C1 proteins (or AU-BP), snRNP U1 70 kDa protein, La protein, Ro protein, and several other RNA-binding proteins. Recently it has been demonstrated that the RNA-binding domain located at the N-terminal part of the protein has a high affinity for a specifically structured RNA element present in the 18S ribosomal RNA. On the other hand the C-terminal part of eIF4B has been also shown to possess an RNA-binding activity but this being sequence-nonspecific. Hence, while the C-terminal nonspecific RNA-binding domain may play an important role in the interaction of the helicase complex with mRNA, the N-terminal domain can be responsible for the binding of eIF4B or the whole helicase complex to initiating 40S ribosomal subunit.

In the experiments with binding to mRNA, eIF4B is capable of releasing the pre-bound cap-binding complex from mRNA. This may suggest that after the cap recognition step has been performed eIF4B destabilizes the retention of eIF4F on mRNA; at the same time it combines with eIF4A and initiates the helicase reaction catalyzed by eIF4A. It has been reported also that eIF4B has a preference to AUG, and to a lesser extent to GUG, over other RNA sequences. Hence, recognizing eIF4F and initiation codon, eIF4B may contribute to specifying the sites on mRNA where the eIF4A-catalyzed helicase reaction to be performed. At the same time, its specific binding to the 40S ribosomal subunit could target the initiating ribosomal particle to the proper unwound region of the mRNA near the initiation codon.

The general model of the sequence of events during the cap binding and the cap-initiated 5'-UTR unwinding looks like as follows (Fig. 15.13). (1) The eIF4E subunit of the cap-binding complex eIF4F binds to the cap-structure in some reversible way. (2) eIF4B forms the helicase complex with eIF4F and thus induces the helicase activity of the eIF4A subunit. The cap-binding complex (its eIF4G subunit) may spread over the cap-adjacent unwound mRNA sequence. (3) In the presence of eIF4B, however, the cap-binding complex is labilely associated with mRNA and tends to be displaced. (4) Free eIF4A interacts now with eIF4B and unwinds the downstream section of mRNA. (5) The repeating acts of interaction of the mRNA-bound eIF4B with eIF4A:ATP progressively unwind the downstream sequence. The cycles, consisting of ATP-dependent eIF4A binding, RNA-induced ATP cleavage and eIF4A release, maintain the single-stranded state of the 5'-UTR of mRNA. (6) Ribosomal initiation 43S complex binds to the unwound mRNA segment including initiation AUG codon, thus becoming initiation 48S complex.

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