Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Abstract

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12) 2 (Ph-L12) 2 (Ph-L12) 2 was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.The ribosomal stalk proteins at the "GTPase-associated center" in the large subunits play a central role in the interaction between the ribosomes and GTP-bound translation factors (1-4). In bacteria, the stalk protein is L7/L12 and is usually present in four copies in the form of two stable dimers bound to the C-terminal regions of L10 (5, 6). The L10⅐L7/L12 complex, together with another protein, L11, binds to the highly conserved domain around 1070 (Escherichia coli numbering is used throughout) of 23 S rRNA through the interaction with the L10 moiety (7,8). The protein complex constitutes a highly flexible domain in the ribosome (9 -15), and the detailed structure within the ribosome has not been resolved by x-ray crystallography (16 -21). It has been found that an E. coli ribosome mutant carrying only one L7/L12 dimer retains significant translation activity (6), and more recently, that there are a few bacterial species whose ribosomes have three L7/L12 dimers in a heptameric complex, L10(L7/L12) 2 (L7/L12) 2 (L7/L12) 2 (22, 23). These findings therefore bring the question as to the significance of multi-stalk dimers in the ribosome into focus.In eukaryotic ribosomes, two related phosphoproteins P1 and P2 are counterparts of bacterial L7/L12 (24 -26). They form heterodimers (27-29), and the two P1-P2 dimers bind to neighboring sites within the C-terminal half of the L10-like stalk base protein P0 (30 -32). This pentameric P0⅐P1-P2 stalk complex can be substituted for E. coli L10⅐L7/L12 complex in the 50 S subunit and makes the ribosome accessible to eukaryotic elongation factors (33)....

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

“…The high copy number does not correlate with thermophilicity, in contrast to suggestions based on analysis of a limited number of bacterial 14,21 . Multiple copies of the protein may increase the encounter frequency with translation factors, thereby facilitating their recruitment to the ribosome.…”

“…Our quantitative analysis of purified ribosomes by mass spectrometry suggests that essentially all potential binding segments on L10 are occupied by L12, which is consistent with the earlier immunoblot analysis of other bacterial ribosomes, for example, from E. coli, T. maritima 9 and M. smegmatis ARTICLE (unpublished data), and mass spectroscopy data 14,21 . This suggests that the maximum possible L12:L10 occupancy is sustained in the bacterial cell.…”

“…Variations in the L12 sequence between species provide the specificity for the interactions with translation factors: the replacement of the L12-L10 stalk in Escherichia coli ribosomes with its eukaryotic counterpart (P0/P1/P2 stalk) makes the ribosome to bind eukaryotic, rather than bacterial elongation factor 13 . The evolutionary and functional advantage of having multiple L12 copies is not known; reducing the L12 copy number to just two copies affects both translation efficiency 14,15 and accuracy 16 .…”

“…Thus, secondary structure predictions can be used to locate helix a8 and estimate the number of potential L12-binding segments. This structure-guided approach was first used to predict the 6:1 ratio of L12:L10 in ribosomes from T. maritima 9 and then to analyse bacterial, archaeal and eukaryotic stalks 14,25 . Sequence analyses of 28 bacterial, species suggested 4:1 and 6:1 stoichiometries in bacteria 14 , with a tendency towards 6:1 for thermophilic organisms, which may, however, be due to the limited number of sequences studied.…”

Mass Spectrometry Defines the Stoichiometry of Ribosomal Stalk Complexes across the Phylogenetic Tree

“…Alignment of the archaeal L10 protein sequences of M. vannielii, M. maripaludis, and M. barkeri with that of the archaeal hyperthermophile Pyrococcus horikoshii (26) (OGT of 98°C) (supplemental Fig. 1) reported during our investigations (27) suggests that the stoichiometry of all stalk complexes would be 6:1. On the other hand the stoichiometry of the M. vannielii stalk complex estimated by scanning of SDS-PAGE gels FIG.…”