Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0.

Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)2 that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)2(aP1)2(aP1)2 complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0·aP1 variant trimers, composed of an aP0 mutant and a single (aP1)2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III, individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0·aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0·aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism.

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α.

In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called 'stalk'. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.

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

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.