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. 2005 Mar 23;33(6):1804-12.
doi: 10.1093/nar/gki321. Print 2005.

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

Nadia Pedullà  1 , Rocco Palermo, David Hasenöhrl, Udo Bläsi, Piero Cammarano, Paola Londei
Affiliations

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

Nadia Pedullà et al. Nucleic Acids Res. .

Abstract

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the beta-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

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Figures

Figure 1
Figure 1
Purification of the recombinant a/eIF2 subunits. The positions of the purified α-, β- and γ-subunits in the Coomassie blue stained SDS–polyacrylamide gel are shown by arrows. The molecular weight markers are (from top to bottom): 90, 67, 45, 30, 21 and 14 kDa.
Figure 2
Figure 2
Reconstitution of the trimeric a/eIF2 with purified subunits. The α-, β- and γ- polypeptides were mixed in equimolar amounts (50 pmol each) and incubated at 65°C for 15 min. The individual subunits and mixtures thereof were electrophoresed on non-denaturing polyacylamide gels (see Materials and Methods). It should be noted that in the native gel system the more basic α-subunits (PI 9.2; 28 kDa) moved slightly ahead of the β-subunits (Isoelectric point 8.1; 15 kDa) despite the difference in size. Migration of the proteins from the cathode towards the anode is depicted on the right-hand side.
Figure 3
Figure 3
GDP and GTP binding by a/eIF2. The a/eIF2, the individual subunits and combinations thereof were incubated with [3H]GDP (top) or [32P]GTP (bottom) and the amount of radiolabelled nucleotide retained by the proteins was determined by filtration through nitrocellulose filters. Closed squares, trimeric a/eIF2; closed circles, β–γ dimer; closed triangles, α–γ dimer; closed diamonds, γ-subunit; and open circles, α-subunit.
Figure 4
Figure 4
Guanine nucleotide exchange on trimeric a/eIF2 and the γ-subunit. The proteins were saturated with [3H]GDP for 15 min at 65°C. Then, a 100-fold excess of cold GDP (closed symbols) or GTP (open symbols) was added and incubation was continued at 80°C. Samples were withdrawn at the indicated times. Squares, trimeric a/eIF2; and circles, γ-subunit.
Figure 5
Figure 5
(A) Interaction of [35S]Met-tRNAi with a/eIF2 subunits, different dimeric complexes and the trimeric a/eIF2 at 70°C. For details see Materials and Methods. Closed circles, complete trimer; closed squares, α–γ dimer; open circles, α–β dimer; open squares, β–γ dimer; closed diamonds, α-subunit; closed triangles, γ-subunit; and crosses, β-subunit. (B) Protection of Met-tRNAi from spontaneous hydrolysis by a/eIF2. [35S]Met-RNAi was incubated with a/eIF2 in the presence of GTP, GDP or Gpp(NH)p and the retained radioactivity was determined by filter binding assays. closed diamonds, 1 mM GTP without a/eIF2 added; open circles, 2 μM a/eIF2 and 1 mM GDP; closed squares, 0.2 μM a/eIF2 and 1 mM GTP; closed triangles, 0.5 μM a/eIF2 and 1 mM GTP; crosses, 1 μM a/eIF2 and 1 mM GTP; closed circles, 2 μM a/eIF2 and 1 mM GTP; and open squares, 2 μM a/eIF2 and 1 mM Gpp(NH)p.
Figure 6
Figure 6
The γ-subunit of a/e IF2 interacts with the 30S subunit. Left panel: each a/eIF2 subunit was incubated with 70S ribosomes and the complexes were separated by non-denaturing PAGE. Western blotting with anti-His tag antibodies was used to visualize the proteins in the gel. Right panel: position of the ribosomal subunits as determined separately by Coomassie blue staining.
Figure 7
Figure 7
Stimulation of M-tRNAi binding to ribosomes by a/eIF2: 30 pmol of 70S ribosomes were incubated with [35S]Met-tRNAi and increasing amounts of a/eIF2 (complete trimer, α–γ dimer or the γ-subunit only). The samples were analysed on a non-denaturing polyacrylamide gel (left panel). Lane 1, [35S]Met-tRNAi alone; lane 2, 70S ribosomes were incubated with [35S]Met-tRNAi without a/eIF2; lanes 3–5, 70S ribosomes were incubated with [35S]Met-tRNAi in the presence of 50 (lane 3), 100 (lane 4) and 150 (lane 5) pmol of the γ-subunit; lanes 6–8, 70S ribosomes were incubated with [35S]Met-tRNAi in the presence of 50 (lane 6), 100 (lane 6) and 150 (lane 8) pmol of the α–γ dimer; and lanes 9–11, 70S ribosomes were incubated with [35S]Met-tRNAi in the presence of 50 (lane 9), 100 (lane 10) and 150 (lane 11) pmol of the trimer. The position of the 30S ribosomal subunit (arrow) was determined separately by Coomassie blue staining. The amount of tRNA bound to ribosomes was quantified using an Instant Imager apparatus (right panel). The total amount of radioactivity in each lane is somewhat variable owing to deacylation undergone by free Met-tRNAi during the run. Circles, complete trimer; triangles: α–γ dimer; and squares, γ-subunit.
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