Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation

Abstract

eIF5 stimulates the GTPase activity of eIF2 bound to Met‐tRNAiMet, and its C‐terminal domain (eIF5‐CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met‐tRNAiMet. The tif5‐7A mutation in eIF5‐CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met‐tRNAiMet and mRNA to 40S subunits in vitro. Interestingly, eIF5‐CTD bound simultaneously to the eIF4G subunit of the cap‐binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5‐7A eliminated eIF5 as a stable component of the pre‐initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate‐limiting defect in this mutant. We propose that eIF5‐CTD stimulates binding of Met‐tRNAiMet and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

“…Two key observations changed this view: (i) the fact that eIF5 plays a role in AUG codon selection, as shown by eIF5-NTD mutations relaxing the stringency of start codon selection (Sui À phenotype) in yeast (Huang et al, 1997); and (ii) the identification of a multifactor eIF5/3/1/TC complex (Asano et al, 2000). Subsequent work has established that eIF5-CTD makes critical contacts with eIF1, eIF2, eIF3, and eIF4G (Asano et al, 2001;Singh et al, 2004aSingh et al, , 2005Yamamoto et al, 2005). A current translation initiation model therefore depicts an early association of TC with eIF5 to form MFC, as shown by solid lines in Figure 7.…”

“…KAY35 (TIF5-FL) (A, C) and KAY39 (hc TIF5-FL) (B, D) were grown in YPD at 301C, treated with cycloheximide for 5 min, and then harvested for preparation of WCE, as described previously (Asano et al, 2001). Twenty five A 260 units of the WCE were fractionated on 5-45% sucrose gradients by centrifugation at 39 000 r.p.m.…”

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

“…COS-7 cells, human embryonic kidney (HEK)293 cells, and normal human fetal lung fibroblasts TIG-7 were grown in DMEM supplemented with 10% FBS. For synchronization experiments, logarithmically growing cells were starved in 0.2% FBS for 48 h and then cultured in fresh media containing 10% FBS for an additional [16][17][18][19][20] h to obtain cell populations enriched in S phase. Alternatively, cells were arrested in prometaphase by adding 50 ng͞ml nocodazole to the medium.…”

“…Lysates were clarified by centrifugation at 23,000 ϫ g for 30 min at 4°C to obtain the supernatant fractions with protein concentrations between 2 and 4 mg͞ml (20). The sample was loaded on a 5-40% sucrose gradient prepared in a buffer containing 20 mM Tris⅐HCl, pH 7.5; 100 mM KCl; and 1 mM MgCl 2 , and centrifuged at 250,000 ϫ g (SW55Ti, Beckman) for 4.5 h at 4°C.…”

Starting the protein synthesis machine: eukaryotic translation initiation

“…To date, it is not known which subunit of eIF3 mediates this interaction and binding of eIF3 to eIF4G has not been found in yeast. Other interactions may act as alternative or additional links between eIF3 and the mRNA: yeast eIF5 can bind to eIF4G (25) and eIF4B interacts with eIF3 subunits in mammals (26) and yeast. (27) eIF4G is a subunit of the heterotrimeric eIF4F complex that binds to the cap structure at the 5 0 end of the mRNA.…”