Unusual Features of the Unusual Ribosomal Elongation Factor EF4 (LepA)

Bacteria, mitochondria, and chloroplasts contain the highly conserved elongation factor EF4; they have in common that the intracellular ionic strength can change dramatically, in contrast to that of archaea and eukaryotic cytoplasm, where EF4 is absent. This factor has the unique function of back-translocating ribosomes, viz., it exerts the opposite function as EF-G. Under unfavorable growth conditions which increase the intracellular ionic strength, such as high salts, low pH, or low temperature, wild-type cells effectively overgrow strains lacking the EF4 gene lepA. Under these conditions most of EF4 is present in the cytoplasm, and only small amounts in the membrane fraction; the opposite is true under optimal conditions, indicating that the membrane is a storage organ for EF4. This factor tunes bacteria for fitness at high ionic strength by (i) improving the active fraction by reactivating unscheduled stalled ribosomes, and (ii) increasing significantly the rate of protein synthesis.     

Fuzzy sets-based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles

Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens. The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data. A number of partitional clustering algorithms and an extensive set of cluster-validity functionals (some already reported and some newly developed) have been applied to a test-data set and to two real-data sets of images. One of the real-data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E. coli 70S monosome in the range of overlap views. These two latter sets had been previously studied by another clustering methodology. The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.     

Polymerization of alpha-hydroxy acids by ribosomes

Over 30 years ago, Fahnestock and Rich reported intriguing data showing the capability of the ribosome to polymerize phenyllactic acid. Although the polymerization was initiated and terminated randomly on polyuridic acids, the given data convincingly suggested that the generated polymer was composed of an approximately 7:3 mixture of phenyllactic acid and phenylalanine. Despite the fact that Fahnestock's conclusion was very likely correct, there have been no reports to follow up the ribosome-catalyzed polymerization of alpha-hydroxy acids until very recently. At the end of 2007, we reported messenger RNA (mRNA)-directed polyester synthesis by using the new emerging method of genetic-code reprogramming in which alpha-hydroxy acids with various kinds of side-chains are assigned to arbitrarily chosen codons. In this work, we have achieved the ribosomal synthesis of polyesters with the sequence composition and length in a fully controlled manner according to the sequence of mRNA. This Concept article describes the background of the method development and its application to the synthesis of polyesters.     

Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism

One of the open questions in the origin of life is the spontaneous formation of primitive cell-like compartments from free molecules in solution and membranes. "Metabolism-first" and "replicator-first" theories claim that early catalytic cycles first evolved in solution, and became encapsulated inside lipid vesicles later on. "Compartment-first" theories suggest that metabolism progressively occurred inside compartments. Both views have some weaknesses: the low probability of co-entrapment of several compounds inside the same compartment, and the need to control nutrient uptake and waste release, respectively. By using lipid vesicles as early-cell models, we show that ribosomes, proteins and lipids spontaneously self-organise into cell-like compartments to achieve high internal concentrations, even when starting from dilute solutions. These findings suggest that the assembly of cell-like compartments, despite its low probability of occurrence, is indeed a physically realistic process. The spontaneous achievement of high local concentration might provide a rational account for the origin of primitive cellular metabolism.     

Lys53 of Ribosomal Protein L36AL and the CCA End of a tRNA at the P/E Hybrid Site Are in Close Proximity on the Human Ribosome

Previously we have shown that the CCA end of a P‐tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre‐translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E‐tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl‐tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.     

Types and Functions of Mitoribosome-Specific Ribosomal Proteins across Eukaryotes

Mitochondria are key organelles that combine features inherited from their bacterial endosymbiotic ancestor with traits that arose during eukaryote evolution. These energy producing organelles have retained a genome and fully functional gene expression machineries including specific ribosomes. Recent advances in cryo-electron microscopy have enabled the characterization of a fast-growing number of the low abundant membrane-bound mitochondrial ribosomes. Surprisingly, mitoribosomes were found to be extremely diverse both in terms of structure and composition. Still, all of them drastically increased their number of ribosomal proteins. Interestingly, among the more than 130 novel ribosomal proteins identified to date in mitochondria, most of them are composed of a-helices. Many of them belong to the nuclear encoded super family of helical repeat proteins. Here we review the diversity of functions and the mode of action held by the novel mitoribosome proteins and discuss why these proteins that share similar helical folds were independently recruited by mitoribosomes during evolution in independent eukaryote clades.