Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants C⌬65 and C⌬81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0⅐P1-P2 binding site. The mutant C⌬107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants N⌬21-N⌬92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants N⌬6, N⌬14, and C⌬18-C⌬81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10⅐L7/L12 complex and L11. Analysis of incorporation of 32 P-labeled P1/P2 into the 50 S subunits with WT-P0 and C⌬81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but C⌬81 bound only one copy each. The hybrid ribosome with C⌬81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.The ribosomal large subunits from all organisms contain an active site termed the "GTPase-associated center" that is responsible for the GTPase-related events in protein biosynthesis. This active site is composed of the two highly conserved domains around 1070 and 2660 (Escherichia coli numbering is used throughout) of 23 S/28 S rRNA and the ribosomal proteins bound to the 1070 region (1-3). The protein components of this site in prokaryotic ribosomes constitute a characteristic pentameric complex, L10(L7/L12) 2 (L7/L12) 2 (4, 5), in which two L7/L12 homodimers bind to the C-terminal regions of L10 (6) and constitute a highly flexible and functionally important lateral protuberance, the so-called "stalk" (7). Although the ribosomal stalk is observed by cryo-electron microscopy (8), the detailed structure of this pentameric complex has not been resolved by x-ray crystallography of ribosomes (9 -11). The chemical features of protein-protein and proteinrRNA interactions in the GTPase-associated center remain to be clarified.The animal ribosomal phosphoproteins P0 and P1/P2 (P proteins) are counterparts of prokaryotic L10 and L7/L12, respectively, although P1 and P2 are related but different proteins, unlike prokaryotic L7/L12 (12-14). In yeast cells, there are two P1-type proteins, P1␣ and P1␤, and two P2-type proteins, P2␣ and P2␤ (15). It is believed that P proteins constitute a pentameric complex, designated here as...

Parameters often are tuned with metabolite concentration time series data to build a dynamic model of metabolism. However, such tuning may reduce the extrapolation ability (generalization capability) of the model. In this study, we determined detailed kinetic parameters of three purified Escherichia coli glycolytic enzymes using the initial velocity method for individual enzymes; i.e., the parameters were determined independently from metabolite concentration time series data. The metabolite concentration time series calculated by the model using the parameters matched the experimental data obtained in an actual multienzyme system consisting of the three purified E. coli glycolytic enzymes. Thus, the results indicate that kinetic parameters can be determined without using an undesirable tuning process.

SummaryAutoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAbinduced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

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