In addition to its role in Met-tRNAMet recruitment, eIF3 also stimulates mRNA binding to the 40S subunit in mammalian and yeast extracts [29, 37, 133, 163]. Because TC binding stimulates mRNA binding to the 40S ribosome [37, 133], eIF3 could act indirectly through its role in TC recruitment. However, eIF3 also seems to have an additional function in mRNA binding independent of TC . The latter is generally attributed to interactions between eIF3 and the mRNA-associated factors eIF4G  or eIF4B . Whereas mammalian eIF4B interacts directly with the eIF3a/ p170 subunit , the yeast homolog of eIF4B (encoded by TIF3) interacts with yeast TIF35/eIF3g  (Fig. 7.2-9F). Mammalian eIF3 contains three subunits that can bind RNA as isolated proteins (eIF3a/p170, eIF3d/p66, and eIF3g/p44) [144, 175-178] (Table 7.2-2) and thus eIF3 could interact directly with mRNA in the initiation complex. Indeed, the b, c, and d subunits of mammalian eIF3 were found crosslinked to globin mRNA in 48S preinitiation complexes . RNA-binding activities of certain eIF3 subunits could mediate direct interactions with the 18S rRNA, as suggested by UV-crosslinking experiments for human eIF3d/p66 . Deletion of the RRM from yeast eIF3g/TIF35 was not lethal but produced a Slg-phenotype. The nature of the RNA that interacts with this RRM is unknown.
Mammalian eIF3 can bind to the hepatitis C virus (HCV) and classical swine fever virus IRES elements, and the eIF3a/p170, eIF3b/p116, eIF3d/p66 and eIF3f/p47 subunits were found crosslinked to these mRNA sequences [180, 181]. The binding region for eIF3 in the HCV IRES has been localized to domains IIIa-b [180, 182] and the cryo-EM map of the IRES-40S complex places this domain extending from the platform side of the 40S subunit just below the mid-line of the particle . This location is consistent with the binding site for eIF3 on 40S subunits visualized in three-dimensional (3D) reconstructions of electron micrographs of negatively stained native 40S subunits [31, 184]; however, eIF3 also makes extensive contacts with the solvent side of the 40S subunit in the model of Lutsch et al . It is unclear whether conventional mRNAs translated by the scanning mechanism will interact with eIF3 in the same manner utilized by the HCV IRES, as the latter bypasses the requirement for the eIF4 factors in forming the 48S complex .
7.2 Mechanism and Regulation of Protein Synthesis Initiation in Eukaryotes | 275 Binding of eIF3 to the 40S Ribosome
Recently, domains in eIF3 required for binding to 40S ribosomes were identified by investigating whether the MFCs formed by mutant versions of TIF32/a and NIP1/c, many of which lack numerous MFC components, can compete with native MFC for stable 40S binding in vivo. The results showed that the N-terminal half of TIF32, NIP1 and eIF5 comprise a minimal 40S binding unit (MBU) sufficient for 40S binding in vivo and in vitro. The N- and C-termini of NIP1 and the TIF32-NTD were required for 40S binding by otherwise intact MFC complexes (TIF32-A8 mutation, Fig. 7.2-9B; NIP1-AB', Fig. 7.2-9C), suggesting that these eIF3 segments make direct contact with the 40S ribosome. Consistently, the TIF32-NTD interacted specifically with 40S subunit proteins RPS0A and RPS10A, and NIP1 interacted with RPS0A and 18S rRNA in vitro. The NIP1-NTD may also contact the 40S subunit in addition to its role in tethering eIF5 to the MFC. eIF5 was necessary for 40S binding only when the TIF32-CTD was absent. Thus, whereas the tif5-7A mutation did not reduce 40S binding by any MFC components except eIF5, it reduced binding by the mutant subcomplexes formed by the C-terminally truncated proteins TIF32-^6 (lacking only eIF2) and TIF32-^5 (^5 and ^6; Fig. 7.2-9B). Interestingly, a 140 nt segment of domain I in rRNA, encompassing helices 16-18, is necessary and sufficient for specific binding of 18S rRNA to the TIF32-CTD in vitro. Hence, the 40S binding activity of the TIF32-CTD may involve direct interaction with domain I of rRNA .
In the cryo-EM model of the yeast 40S subunit , RPS0A is on the solvent side of the 40S subunit between the protuberance (pt) and beak (bk). Hence, binding of the TIF32-NTD and NIP1 to RPS0A would place this portion of eIF3 on the solvent side of the subunit, consistent with the EM analyses of 40S-eIF3 complexes [31, 184] and the location of the HCV IRES (and its eIF3-binding domain) on the 40S subunit [180, 182]. Interaction between the TIF32-CTD and helices 16 and 18 of the rRNA would provide eIF3 with access to the 60S-interface side, as these helices are accessible from both sides of the 40S subunit. It was proposed that the bulk of eIF3 would bind to the solvent side of the 40S whereas the TIF32-CTD and NIP1-NTD would wrap around helix 16 or penetrate the cleft between the beak (bk) and shoulder (sh), respectively, gaining access to the interface side of the subunit. The P-site is located on the interface side ~50-55 Á from the binding sites for TIF32-CTD and NIP1-NTD predicted in this model . This separation is comparable with the dimensions of the ^-subunit of eIF2 , making it reasonable to propose that the NTD of eIF2^ can remain connected to the TIF32-CTD and the NIP1-NTD/eIF5 subassembly of the MFC while Met-tRNAMet is bound to the P-site. In contrast, the connections between eIF1 and the TIF32-CTD and NIP1-NTD might have to be severed to allow eIF1 to bind near the P-site .
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