Multiple aminoacyl-tRNA synthetases (translases) in temperature acclimation of eurythermal fish

The temperature dependency of activity of the entire set of aminoacyl-tRNA synthetases (protein synthetic translases) has been studied in the laboratory rat and in toadfish (Opsanus tau) acclimated to 20 δC or to 10 δC. The complex temperature responses of these enzyme systems reveal the presence of multiple forms for the translases of most amino acids and show adaptive behavior of these systems with respect to body temperature of the animal. The phenylalanine translase system has been studied in detail, and adaptation of this system at low temperatures correlates with adaptation in the elongation factor system. All known protein synthetic components appear to be coordinated in adaptive responses with the exception of ribosomes. Our data indicate no rôle for ribosomes in adaptation of the protein synthetic system and apparently no rôle for ribosomes in protein synthesis at all in rat and fish. This finding may solve some long-standing paradoxes in the protein synthesis field concerning the mechanism by which ribosomes participate in protein synthesis.     

Ribosomal crystallography: peptide bond formation and its inhibition

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.     

Protein deficient chloroplast ribosomes in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analyzed and compared by two-dimensional gel electrophoresis. The mixothrophically grown yellow-76 mutant differs from wild-type cells in lowered chlorophyll content and photosynthetic activity per chlorophyll unit. The latter is connected with the decreased activity of the ribulose-I,5-diphosphate-carboxylase enzyme. Analytical ultracentrifugation of cell extracts shows a normal amount of free 70S ribosomes and 50S subunit in the mutant cells. Two-dimensional gel electrophoresis shows considerable alterations in the protein composition of the 70S ribosomes of the mutant. Two proteins are absent from the electrophoretograms of the yellow-76 mutant, and seven proteins are present in reduced amounts. The genetical analysis shows a Mendelian pattern of inheritance, indicating that protein alterations presumably are localized in nuclear DNA.Abbreviation MNNG N-methyl-N-nitro-N-nitrosoguanidine     

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.