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. 2010 Aug 27;285(35):26779-26787.
doi: 10.1074/jbc.M110.119693. Epub 2010 Jun 21.

GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor

Sergey E Dmitriev  1 , Ilya M Terenin  1 , Dmitri E Andreev  1 , Pavel A Ivanov  2 , Jacov E Dunaevsky  1 , William C Merrick  3 , Ivan N Shatsky  4
Affiliations

GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor

Sergey E Dmitriev et al. J Biol Chem. .

Abstract

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.

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Figures

FIGURE 1.
FIGURE 1.
Reconstitution of 40 S preinitiation complexes on the HCV IRES using a partially purified RSW fraction of RRL containing eIF2A and GTP-independent Met-tRNA binding activity and detection of the complexes by toe-printing (lanes 1–7) or by RelE printing (lanes 8–11). Position of the AUG start codon is indicated. The bands originating from the binary HCV IRES × 40 S complex and those originating from the HCV IRES × 40 S × Met-tRNAiMet ×eIF2 (eIF2D) complexes are denoted on the right of the gel as 40 S and 48 S, respectively. Sequencing lanes obtained for the corresponding cDNA using the same primer are presented on the left.
FIGURE 2.
FIGURE 2.
Purification of the GTP-independent Met-tRNA binding activity. A, scheme of purification ((NH4)SO4 precipitation is abbreviated as ASP. B, Coomassie-stained gel that exemplifies two fractions (15 and 16) of HeLa RSW from the last step of purification of GTP-independent Met-tRNAiMet binding activity on Mono Q, of which fraction 16 had a much higher activity. Positions of two bands (∼70 and ∼100 kDa), with which this activity could be associated, are shown with filled and open arrowheads, respectively. M denotes position of standard molecular weight markers. C, upper part, conserved domains identified in the eIF2D sequence; bottom part, alignment of SUI1 domains of eIF2D and translation initiation factor eIF1. cd00474, consensus sequence of SUI1 domain from Conserved Domain Data base.
FIGURE 3.
FIGURE 3.
Functional properties of eIF2D. A, lanes 1–7 show experiments analogous to those presented in Fig. 1A, but the purified recombinant factor was used instead of partially purified RSW fraction. Lanes 8–11 analyze the contribution of aminoacyl moiety of Met-tRNAiMet to its binding with 40 S ribosomal subunits. The HCV-UUU mRNA represents the HCV IRES where the initiation codon was mutated from AUG to UUU to test binding of cognate tRNAPhe in the presence of eIF2D (lanes 12–14). B, membrane binding assay. Recombinant eIF2D was tested for activity in [35S]Met-tRNAiMet binding with 40 S ribosomal subunits programmed with the AUG triplet. Bkg, background (no tRNA); control, no eIF2D added; eIF2A-1 and eIF2A-2 are two forms of eIF2A (see the text) purified to homogeneity.
FIGURE 4.
FIGURE 4.
Effect of eIF2D on the formation of 48 S preinitiation complexes with various model constructs. A, assembly of the complexes with the leaderless mRNA construct cIlacZ and its derivatives. cIlacZ is an mRNA with the 5′-terminal gAUG sequence described by Andreev et al. (13). 5A-cI and 5G-cI are cIlacZ derivatives with GAAAA and GGGCC sequences added to its 5′ end, respectively, described in the same paper. cI-GUG is a leaderless cIlacZ construct where the 5′-terminal AUG was replaced by GUG. Other designations are as in Figs. 1 and 3. B, formation of the 48 S complex on the CAA-cIlacZ mRNA, which represents the sequence (CAA)19 added to the 5′ end of cIlacZ.
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