sufficient for efficient ribosomal attachment to capped mRNA (2). This fragment of eIF4G binds both eIF4E and eIF4A and probably coordinates their activities so that a cap-proximal region of mRNA is unwound and is thus rendered accessible to an incoming 43S complex so it can bind productively. The molecular interactions that enable the incoming 43S complex to bind this “prepared” template are not known but are thought to involve interaction of the eIF3 component of 43S complexes with cap-associated eIF4G. The bound ribosomal “complex I” was arrested in a cap-proximal position and did not reach the initiation codon (Fig. 1 A).
Two additional activities present in rabbit reticulocyte lysate (RRL) enabled 43S complexes to reach the initiation codon, forming “complex II” without being arrested at the initial binding site (Fig. 1 B). These small factors were purified and identified by sequencing as eIF1 (13.5 kDa) and eIF1A (19 kDa) and could be functionally replaced by corresponding recombinant polypeptides. These two factors acted synergistically; eIF1 A without eIF1 enhanced eIF4F-mediated binding of 43S complexes to mRNA but did not enable these complexes to reach the initiation codon, whereas eIF1 without eIF1A reduced the prominence of the cap-proximal complex I and promoted formation of low levels of 48S complexes. The interaction with mRNA of 48S complexes assembled in the absence of eIF1A differed subtly from complexes formed in their presence, in that only two (+16–+17) rather than three toeprints (+15–+17) were apparent. eIF1A therefore increases the competence of 43S complexes to bind mRNA and the processivity of scanning 43S/eIF1/mRNA complexes. eIF1A also stabilizes binding of the ternary complex to 40S subunits in the absence of mRNA (3, 4), presumably by an allosteric mechanism, because it is not known to interact directly with any component of the ternary complex. This stabilization by eIF1A is weak but might be indicative of a role for eIF1A in ensuring that initiator tRNA and mRNA adopt the correct relative orientation on the scanning ribosomal complex.
eIF1A comprises an oligonucleotide-binding (OB) ß-barrel fold that closely resembles prokaryotic initiation factor IF1 (and corresponds to the region of sequence homology between them) and an additional C-terminal domain (4). The experimentally determined RNA-binding surface of eIF1A is large, extending over the OB fold and the adjacent groove leading to the second domain. Mutations at multiple positions on this surface resulted in a reduced ability of eIF1A to promote assembly of 48S initiation complexes at the initiation codon. The RNA ligand for eIF1A is not known, but by analogy with IF1 (5), eIF1A might bind 18S ribosomal RNA in the ribosomal A site.
In the absence of eIF1 and eIF1A, the mRNA-binding cleft on 40S subunits appears to be open, because they can bind mRNA in an end-independent manner during initiation by internal ribosomal entry (see below). eIF1 and eIF1 A may contribute to the correct interactions of components of the 43S complex with mRNA that enable it to enter a processive mode, for example by closing this cleft directly or indirectly and possibly even by forming part of the channel on the 40S subunit through which mRNA moves during ribosomal scanning.
Experiments done by using competitor mRNAs indicated that complex I cannot be “chased” directly into complex II and is therefore not its immediate precursor. Complex I is aberrantly assembled (because it is arrested at a non-AUG triplet and is unable to scan to the initiation codon) and is intrinsically unstable. eIF1 and eIF1A together (but not individually) promote dissociation of complex I and enable the released 43S complex to rebind mRNA in a competent state to scan to the initiation codon (Fig. 1 C). eIF1 alone is able to recognize and destabilize ribosomal complexes incorrectly assembled by internal ribosomal entry (see below). Identification of this activity of mammalian eIF1 is consistent with characterization of its yeast homologue Sui1 as a monitor of translation accuracy. Mutations in Sui1 allow aberrant initiation in vivo at non-AUG codons by mismatch base pairing with Met-tRNAi (e.g., ref. 6). Determination of the solution structure of eIF1 by NMR (7) has revealed that these mutated residues form part of a surface that is almost perfectly conserved among all eIF1 homologues and that is likely directly involved in initiation codon selection by eIF1.
In summary, we have determined the set of factors required for binding of a 43S complex to a model native capped mRNA and for it to scan to the initiation codon. These experiments were done by using ß-globin mRNA, and it is possible that ribosomal scanning on longer or more highly structured 5' NTRs may require additional as-yet-unidentified factors, for example to enhance processivity or to promote unwinding of stable secondary structures. Almost all aspects of the mechanism of ribosomal scanning remain uncharacterized (8). For example, scanning is an ATP-dependent process, but it is not known whether ribosomal movement itself involves hydrolysis of ATP or whether chemical energy is required only to unwind secondary structure in the 5' NTR to permit ribosomal movement by one-dimensional diffusion from its initial 5'-terminal attachment site. The ability to reconstitute this process in vitro will enable this and other outstanding questions to be addressed.
Factor Displacement from the 48S Complex and Joining to a 60S Subunit to Form Active 80S Ribosomes. The 48S complex assembled at the initiation codon of ß-globin mRNA is bound by factors that must be displaced before the 40S subunit/mRNA/Met-