Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

Abstract

Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0(aP1)(2)(aP1)(2)(aP1)(2), can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.

Fig. 1.

Binding of the archaeal stalk protein aP1 to elongation factor aEF-2. In the presence of excess amounts (1 mM) of GDP (A) or GMPPCP (B), aP1 homodimer (200 pmol) was incubated without aEF-2 (lane 1) or with 200 pmol (lane 2), 400 pmol (lane 3), 600 pmol (lane 4), or 800 pmol (lane 5) of aEF-2 in 10 μL solution at 70 °C. aEF-2 (200 pmol) was also incubated alone (lane 6). Individual samples were subjected to a gel mobility shift assay, as described in Materials and Methods. In the presence of the same amounts of GDP (C) and GMPPCP (D) as in A and B, the complexes were formed by mixing 200 pmol of aP1 dimer and 600 pmol of aEF-2 (lane 2), in the presence of 1 nmol (lane 3), 2 nmol (lane 4), or 4 nmol (lane 5) of the peptide that comprised the C-terminal 18 amino acids of aP1. Gel analysis was as in A and B.

Fig. 2.

Analyses of aP1•aEF-2 binding using the mutant proteins. (A and B) Homodimers of the aP1 point mutants, Leu106Ser (A) and Phe107Ser (B) (200 pmol each), were incubated without aEF-2 (lane 1) or with 400 pmol (lane 2), 800 pmol (lane 3), 1.2 nmol (lane 4), or 1.6 nmol (lane 5) of aEF-2. aEF-2 (400 pmol) was also incubated alone (lane 6). (C) 200 pmol of wild-type aP1 were incubated with domain I of aEF-2 as in A and B. Gel analysis was carried out as in Fig. 1.

Fig. 3.

Binding of the aP1 stalk protein to GTPase factors other than aEF-2. (A–C) aP1 homodimer (200 pmol) was incubated without factor (lane 1) or with 400 pmol (lane 2), 800 pmol (lane 3), 1.2 nmol (lane 4), 1.6 nmol (lane 5), or 2.0 nmol (lane 6) of aEF-1α (A), aIF5B (B), or aIF2γ (C). aEF-1α, aIF5B, and a aIF2γ (400 pmol each) were also incubated alone (lane 7 of A–C, respectively). (D) The aP1•aEF-1α complex was formed by mixing 200 pmol of aP1 homodimer and 400 pmol of aEF-1α (lane 2), and 10 nmol of the C-terminal peptide of aP1 were also added (lane 3). The aP1•aIF5B complex was formed by mixing 200 pmol of aP1 dimer and 400 pmol of aIF5B (lane 4), and 10 nmol of the peptide were also added (lane 5). Gel analysis was carried out as in Fig. 1.

Fig. 4.

Binding of the P0-P1 stalk complex to multiple molecules of aEF-2. (A) In the presence of GDP (Left) or GMPPCP (Right), the aP0•aP1 heptameric complex (25 pmol) was incubated without aEF-2 (lane 1) or with 25 pmol (lane 2), 50 pmol (lane 3), 100 pmol (lane 4), 200 pmol (lane 5), 250 pmol (lane 6), or 380 pmol (lane 7) of aEF-2 at 70 °C. aEF-2 (25 pmol) was also incubated alone (lane 8). Gel analysis was carried out as in Fig. 1. (B) C(s) distributions from sedimentation velocity analytical ultracentrifugation of the following samples: The aP0•aP1 heptameric complex, which was preincubated without aEF-2 (Top) or with aEF-2 at a molar ratio of 1∶1 (Second Row), 1∶2 (Third Row), 1∶3 (Fourth Row), and 1∶4 (Fifth Row). aEF-2 was also incubated alone (Bottom).

Fig. 5.

Functional effect of the point mutations at the C termini of eukaryotic P0, P1, and P2. The point mutants, Phe315Ser in P0, Phe111Ser in P1, and Phe111Ser in P2, were generated as described in Materials and Methods. The truncation mutants ΔC55-P0, ΔC52-P1, and ΔC50-P2, in which the C-terminal 55, 52, and 50 amino acids were deleted from P0, P1, and P2, respectively, were prepared as described previously (25). The P0•P1•P2 complexes were reconstituted (11) using the mutant proteins indicated below the bars. Each complex (10 pmol) was incorporated into the E. coli 50S core (2.5 pmol) with eL12, and eukaryotic eEF-2-dependent GTPase activity was assayed in the presence of E. coli 30S subunits (11).

Fig. 6.

A schematic representation of the nucleotide-independent interaction between the ribosomal stalk protein aP1 and elongation factor aEF-2. Shown in orange is the C-terminal segment of a single copy of aP1 among the six copies bound to aP0. The C-terminal segment interacts with aEF-2 (blue) that is bound to GTP or GDP. The curved line between the C-terminal segment and the main body of the ribosome represents the flexible hinge region of aP1, which connects the C-terminal segment with the N-terminal domain (9).