The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1) 2 (P1) 2 (P1) 2 ) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factordependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.

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, aP0ðaP1Þ 2 ðaP1Þ 2 ðaP1Þ 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.GTPase-associated center | ribosome protein P0 | ribosome protein P1 | hyperthermophilic archaeon M ajor dynamic steps in translational initiation, elongation, and termination are promoted by the actions of several translational GTPase factors (1-3). During the elongation step, two elongation factors bind alternately to the ribosome in their GTP-bound states. After they have carried out their respective functions, which are linked to GTP hydrolysis, they then dissociate from the ribosome. The large ribosomal subunit contains an active center, termed the GTPase-associated center or factorbinding center, which interacts with the elongation factors (4). The GTPase-associated center plays a crucial role in the recruitment of translation factors, GTP hydrolysis, and the release of inorganic phosphate, which is required for the subsequent dissociation of the factor. Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).In the case of bacteria, the structure and function of the stalk protein L7/L12 (termed L12 hereafter) is well established. The L12 protein is composed of an N-terminal dimerization domain and a globular C-terminal doma...

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