Jean-Paul Reboud

The puzzling lateral flexible stalk of the ribosome

The lateral flexible stalk of the large ribosomal subunit is made of several interacting proteins anchored to a conserved region of the 28S (26S) rRNA termed the GTPase‐associated domain or thiostrepton loop. This structure is demonstrated to adopt puzzling changes of conformation following the different steps of the elongation cycle. Some of these proteins termed the P‐proteins in eukaryotes and L10 and L7/L12 in bacteria, present little structural similarities between Eubacteria on one side and Archae and Eukaryotes on the other side. However, up to now, these proteins seem to present a similar macromolecular organisation and they have been involved in the same functions. Convincing evidence attests that these proteins participate in elongation factor binding to the ribosome, and it has been suggested that these proteins might be evolved in a GTP hydrolysis activating protein activity. Involvement of these proteins in the translational mechanism is discussed. Moreover, in eukaryotes, small P‐proteins are also found as isolated proteins in a cytoplasmic pool that exchanges with the ribosome‐associated P‐proteins. Moreover, a part of the ribosomal proteins is phosphorylated (hence their P‐protein names). The biological signification of these particularities is discussed.

Pivotal role of the P1 N-terminal domain in the assembly of the mammalian ribosomal stalk and in the proteosynthetic activity.

In the 60 S ribosomal subunit, the lateral stalk made of the P-proteins plays a major role in translation. It contains P0, an insoluble protein anchoring P1 and P2 to the ribosome. Here, rat recombinant P0 was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. This method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with P0. Furthermore, the use of truncated mutants of P1 and P2 indicated that residues 1–63 in P1 connected P0 to residues 1–65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with P0 or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant P0 in complex with phosphorylated P2 and either P1 or its (1–63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded P0 was functional and residues 1–63 only in P1 were essential. Furthermore, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.

A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.

Interaction of phosphorylated elongation factor EF‐2 with nucleotides and ribosomes

The intrinsic fluorescence emission spectrum of elongation factor EF‐2 due to the 7 Trp residues was not modified after complete phosphorylation of the factor by the specific Ca2+/Calmodulin‐dependent kinase III. The effect of nucleotide binding on this fluorescence revealed differences between phosphorylated and unmodified EF‐2. Low concentrations of GTP had a smaller quenching effect on the fluorescence of phosphorylated EF‐2 than on the fluorescence of unmodified EF‐2, whereas GDP had exactly the same quenching effect on the fluorescence of both samples. These results suggest that phosphorylation of EF‐2 decreased its affinity for GTP but not for GDP. Ability of phosphorylated EF‐2 to form a ternary complex with ribosomes in the presence of a non‐hydrolysable GTP analog and its ability to protect ribosomes against ricin‐inactivation were both decreased to the same extent. The lower affinity of phosphorylated EF‐2 for GTP could be responsible for a weaker and/or incorrect interaction of the factor with the ribosome, in particular with the ricin‐site of the 28‐S rRNA assumed to be involved in translocation initiation.

Properties of elongation factor-2 fragments obtained by partial proteolysis.

Rat liver elongation factor eEF-2 was treated with endoproteinase Glu-C. Two major fragments were obtained, which were identified by N-terminal sequencing and purified. The larger one (F61) contained 554 residues including the N-terminal end, and after a second cleavage released a N-terminal peptide (F7) of 62 residues. The smaller one (F34) contained the other 303 residues including the C terminal end. F61 and F34, either isolated or after combination, were unable to catalyze protein synthesis. However, we show by fluorimetry that F61 could still interact with GTP and GDP. This fragment was was able to participate into a ternary complex with ribosome and GDP, but not with ribosome and a GTP analogue. It was unable to protect the ribosome against ricin-inactivation and to be phosphorylated by the eEF-2-specific Ca(2+)-calmodulin-dependent kinase, though it contained Trp221 and Thr56 involved in these reactions. On the other hand, F34 could be ADP-ribosylated in the presence of NAD+ and diphtheria toxin, but this fragment was apparently unable to bind to ribosomes. These results and those obtained with other proteinases are discussed in the light of the data published recently which show the existence of five different domains in the three-dimensional structure of EF-G.

Autoantibodies directed against ribosomal proteins in systemic lupus erythematosus and rheumatoid arthritis: a comparative study.

To assess the specificity of autoantibodies (aAbs) directed against the ribosomal P-proteins (RPPaAbs) in patients with systemic lupus erythematosus (SLE) and to investigate aAbs directed to other ribosomal proteins, 100 SLE, 100 rheumatoid arthritis (RA), 25 thyroiditis and 20 blood-donors were analyzed in a comparative study using an immunoblotting technique. Forty-eight percent of SLE sera contained aAbs directed against the ribosomal proteins of the 60 S subunit compared to 9% for RA, 5% for blood donors and 0% for thyroiditis. RPPaAbs were only found in SLE (25%) and aAbs directed to a 31 kDa and/or a 28 kDa protein of the 60 S subunit were found with a statistically higher frequency for SLE compared to RA (p <0.0001). aAbs directed to proteins of the 40 S subunit were present in 63% of the SLE sera compared to 42% for RA, 4% for thyroiditis and 5% for blood donors. The number of positive sera was not statistically different between SLE and RA but a much more intense reactivity was observed for SLE sera. These data shows that the aAbs against the ribosomal proteins, especially the P-proteins along with the 28 and 31 kDa proteins of the 60 S subunit proteins, can be considered as useful biological markers for the diagnosis of SLE in clinical practice.