Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Influence of Abutment Design on Stiffness, Strength, and Failure of Implant-Supported Monolithic Resin Nano Ceramic (RNC) Crowns

BACKGROUND Recent technical development allows the digital manufacturing of monolithic reconstructions with high-performance materials. For implant-supported crowns, the fixation requires an abutment design onto which the reconstruction can be bonded. PURPOSE The aim of this laboratory investigation was to analyze stiffness, strength, and failure modes of implant-supported, computer-assisted design and computer-aided manufacturing (CAD/CAM)-generated resin nano ceramic (RNC) crowns bonded to three different titanium abutments. MATERIALS AND METHODS Eighteen monolithic RNC crowns were produced and loaded in a universal testing machine under quasi-static condition according to DIN ISO 14801. With regard to the type of titanium abutment, three groups were defined: (1) prefabricated cementable standard; (2) CAD/CAM-constructed individualized; and (3) novel prefabricated bonding base. Stiffness and strength were measured and analyzed statistically with Wilcoxon rank sum test. Sections of the specimens were examined microscopically. RESULTS Stiffness demonstrated high stability for all specimens loaded in the physiological loading range with means and standard deviations of 1,579 ± 120 N/mm (group A), 1,733 ± 89 N/mm (group B), and 1,704 ± 162 N/mm (group C). Mean strength of the novel prefabricated bonding base (group C) was 17% lower than of the two other groups. Plastic deformations were detectable for all implant-abutment crown connections. CONCLUSIONS Monolithic implant crowns made of RNC seem to represent a feasible and stable prosthetic construction under laboratory testing conditions with strength higher than the average occlusal force, independent of the different abutment designs used in this investigation.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

Protein Hydrolysis and Nitrogen Remobilisation in Plant Life and Senescence

In plant cells, as in all other cells, proteins are submitted to permanent turnover, and the intracellular content of a given protein depends on its rate of both synthesis and degradation. The life time of most proteins is shorter than that of the cell. Thus, in young leaves of Lemna minor, the average half-life of protein was estimated to be 7 days, and it was shorter under stress conditions (Davies 1982). Such observations mean that nitrogen and amino acid fluxes are both cylic and permanent. Although protein turnover may appear wasteful, in terms of energy, numerous studies have shown that proteolysis provides multiple functions in cell physiology, and is an essential regulatory mechanism of cell metabolism and development.