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Ribosome is a macromolecular complex, which is responsible for protein biosynthesis. Two bacterial ribosomal subunits contain more than 4000 RNA nucleotides and 50 proteins. Ribosome assembly is a complicated multi-step process, vitally important for cell. In this review we summarised present-day conceptions about the mechanism of the bacterial ribosome assembly in the cell and in vitro model systems. Some details of the assembly of this machinery are still-unknown.

Four protein factors, belonging to the GTPase superfamily, participate in bacterial biosynthesis: IF2, EF-G, EF-Tu and RF3. The exact role and mechanism of action of these proteins was of particular interest over the last several decades. Recent advances in structural methods of ribosomal research, especially application of cryoelectron microscopy, provided powerful experimental tools for the investigation of ribosomal dynamics during translation. Simultaneously, progress in the biochemical investigation of translation allowed us to link structural rearrangements occurring in the ribosome to functional changes in the ribosome-bound translational GTPases--GDP/GTP exchange, GTPase activation and its conformational changes. Accumulated data have lead to formulation of current models of mechanisms of translation. More and more facts testify in favor of the idea that the ribosome plays a prominent role both in the nucleotide exchange and in GTPase activation, thus playing the role both of GAP and GEF for RF3, IF2 and EF-G. In our work we attempted to systematize the most important experimental findings and models for mechanisms of GTPases function and regulation in prokaryotic translation.

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their trafficking are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.

Protein biosynthesis is a complex biochemical process. It integrates multiple steps where different translation factors specifically interact with the ribosome in a precisely defined order. Among the translation factors one can find multiple GTP-binding or G-proteins. Their functioning is accompanied by GTP hydrolysis to the GDP and inorganic phosphate ion Pi. Ribosome stimulates the GTPase activity of the translation factors, thus playing a role analogues to GTPase-activating proteins (GAP). Translation factors--GTPases interact with the ribosome at all stages of protein biosynthesis. Initiation factor 2 (IF2) catalyse initiator tRNA binding to the ribosomal P-site and subsequent subunit joining. Elongation factor Tu (EF-Tu) is responsible for the aminoacyl-tRNA binding to the ribosomal A-site, while elongation factor G (EF-G) catalyses translocation of mRNA in the ribosome by one codon, accompanied by tRNA movement between the binding sites. In its turn, release factor 3 (RF3) catalyse dissociation of the ribosomal complex with release factors 1 or 2 (RF1 or RF2) following the peptide release. This review is devoted to the functional peculiarities of translational GTPases as related to other G-proteins. Particularly, to the putative GTPase activation mechanism, structure and functional cycles.

We undertook a search for proteins interacting with protein p45 by the method of two-hybrid screening in order to determine the function of the Sec14p-like protein p45. A screening of the yeast library of rat lung cDNA, six proteins specifically activating the reporter genes of a two-hybrid system and 21 unlikely protein partners of protein p45 were identified. The most likely candidate for the role of a p45 partner is the surfactant protein C (Sftpc). These results and previous studies led us to the hypothesis that protein p45 fulfills its protective function by participating in the biogenesis of cell membranes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Characteristics of G-proteins were studied in renal medulla of rats and mice of different ages. It was established that G-protein alpha-subunit gene expression has a specific dynamics with maximum on the 5-8-th day of life and at the end of weaning. It was shown that vasopressin does not stimulate G-protein GTPase activity in membrane preparations from immature kidneys. The differences of G-protein chromatographic characteristics were revealed. It is suggested that the development of vasopressin sensitivity of the kidney is related with the changes in G-protein system.

Heparin (20-40 micrograms/ml) inhibits by 50% both the ATP hydrolase and the delta pH formation in isolated yeast vacuoles. The GTPase activity is not inhibited under these conditions. ATP prevents this inhibition, being added 10 min before heparin. Heparin suppresses the phosphorylation of vacuolar proteins in vitro. It is supposed that the protein kinase, similar to casein kinase I, participates in the regulation of activity of the vacuolar ATPase.

A hypothesis presented in the paper discusses the role of valine substitution in p21 protein of H-ras, a product of ras family oncogene. The event was followed by a marked decrease in GTPase activity of the protein and alteration of its function in the cell. Recent results of X-ray structural analysis were compared with the model suggested by the authors earlier. The role of valine substitution in sickle-cell anemia held to be a genetic mechanism of protection against malaria is discussed. Based on similarity of valine substitution in p21 protein and hemoglobin, a relationship between certain forms of malignant transformation and malaria at molecular level is suggested.

Functional expression of bovine visual rhodopsin in the cell-free translation system with cotranslational insertion of the protein into phosphatidylcholine liposomes is described. The recombinant rhodopsin has spectral and functional properties similar to those of natural rhodopsin from bovine retina. Two mutant rhodopsins with amino acid substitutions in the hydrophilic C-terminal domain were obtained using oligonucleotide-directed mutagenesis. It was found that substitution Cys-316----Ser does not affect rhodopsin's ability to activate the visual amplification cascade, whereas double mutation Asp-330----Asn, Asp-331----Asn dramatically lowers the rhodopsin functional activity.

The effect of preincubation of preparations of the outer segments of optic rods with the nonhydrolyzed analog GTP-guanilyl-5'-imidodiphosphate (Gpp(NH)p) and NaF, the combined effect of these agents as well as the action of (NH4)2SO4 (10-800 mM), MgSO4 (2-50 mM) and induction of peroxide oxidation of lipids are studied as applied to the catalytic activity of phosphodiesterase of cyclic nucleotides. Gpp(NH)p and NaF are shown to be tightly bound to GTP-binding proteins (G-proteins) of outer segments of optic rods, additional activation of phosphodiesterase in the presence of Gpp(NH)p being observed after preincubation with NaF and subsequent washing of the membrane. A problem on different binding sites of the ion F and Gpp(NH)p on G-proteins is discussed. It is found that (NH4)2SO4 does not affect the basal activity of phosphodiesterase but inhibits the activating effect of Gpp(NH)p and NaF on the enzyme. Induction of peroxide oxidation of lipids prevented by the addition of ionol (antioxidant) in a dose of 5.10(-4) M has the same effect. Changes in the concentration of Mg2+ in the medium influence insignificantly the basal activity of phosphodiesterase but are necessary for manifestation of the activating effect of Gpp(NH)p and NaF.

The innervation of the pial arteries as well as the activity of enzymes (phosphorylase I, II, III, succindehydrogenase, lactate dehydrogenase, ATPase, GTPase and CTPase) responsible for vascular smooth muscle function were studied histochemically on total microscopic preparations of rabbit pia matter. An especially rich adrenergic and cholinergic innervation was found around the active microvascular effectors - sphincters of pial and precortical arterial off-shoots. The nerve fibers followed the radial arteries entering the cerebral cortex. No differences were detected between the pial arteries and active microvascular effectors in the enzyme activity.

The activity of guanosine diphosphatase (GDPase) and guanosine triphosphatase (GTPase) was determined in mitochondrial fractions and postmitochondrial supernatant fluid from the rat brain and liver nuclei 1, 3, 6, 24 and 48 hours after their irradiation by gamma-quanta of [60Co] in a dose of 774 mC/kg. Under the influence of irradiation the activity of GDPase in the liver and brain mitochondria increased, that of GTPase in the liver mitochondria underwent phase changes. The GDPase activity tended to increase in postmitochondrial supernatant fraction of the liver and brain; the GTPase activity was at first inhibited and then increased.