Soluble Protein Factors Research Articles

Import of RNA into Leishmania mitochondria occurs through direct interaction with membrane-bound receptors.

Cytoplasmic tRNAs are imported into the kinetoplast mitochondrion of Leishmania, but the mechanism of import is unknown, particularly whether RNA is transferred as a ribonucleoprotein complex through the protein import pathway or by a distinct receptor-mediated mechanism. Using isolated mitochondria, it was shown that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with high affinity to the mitochondrial surface in the absence of soluble protein factors to form an import intermediate. Two classes of binding site of apparent Kd 0.3 and 10 n, respectively, were distinguished. tRNA from Leishmania, but not yeast, competitively inhibited the binding. Northwestern blot analysis revealed the presence of a 15-kDa RNA binding protein on the mitochondrial surface. Whereas receptor binding was resistant to heparin and KCl, internalization was sensitive to both reagents. These results are consistent with the presence of a direct mechanism of receptor-mediated RNA import on Leishmania mitochondria.

Pyridine-Promoted Factor- and Energy-Free Peptide Synthesis Systems Prepared from Various Organisms Including Prokaryote, Eukaryote, and Mitochondria

We demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus [Nitta et al. (1994) J. Biochem. 115, 803-807; ibid., (1995) 118, 841-849] but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms. Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms. The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another. All these systems required Mg2+, and only mitochondrial system showed the extra Mn(2+)-requirement, which enhanced the yield by several fold. The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80 degrees C. The rationale for these observations remains to be solved.

Template-dependent polypeptide synthesis in a factor- and energy-free translation system promoted by pyridine.

We demonstrate here that a high concentration (40-70%) of pyridine, an aromatic tertiary amine catalyst, is able to promote translation on ribosomes without the presence of soluble protein factors or chemical energy sources. Compared with Monro's fragment reaction [Methods Enzymol. 20, 472-481 (1971)] which reflects only the peptidyltransferase step, this novel translation system can produce polypeptides with chain lengths of at least several tens of residues depending on the template RNA. In the presence of 60% pyridine, poly(U) and poly(UC) promoted incorporation of the respective amino acids, phenylalanine and serine-leucine, twofold, whereas poly(A) promoted the incorporation of lysine by only 25%. The degrees of polymerization of phenylalanine and lysine were up to the decamer and around 40mer, respectively. In poly(UC)-dependent oligo(serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products. This novel pyridine system evidently differs from the non-enzymatic translation system reported by Gavrilova and Spirin [FEBS Lett. 17, 324-326 (1971)]; the former system displays partial resistance toward deproteinization reagents such as SDS and proteinase K, whereas the latter system is completely sensitive.

Basis for Changes in the Auxin-Sensitivity of Avena sativa(Oat) Leaf-Sheath Pulvini during the Gravitropic Response

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

ATPase strongly bound to higher eukaryotic ribosomes.

80 S ribosomes from a number of higher eukaryotic organisms are able to hydrolyse ATP and GTP without the addition of soluble protein factors. ATPase seems to be an intrinsic activity of the ribosome, as indicated by the findings that ATPase activity is not diminished upon dissociation of ribosomes and reassociation of subunits, by washing with 0.66 M (KCl + NH4Cl) or 0.6 M LiCl treatment and ethanol precipitation; 1.5 M LiCl treatment removes only 40% ATPase activity. 80 S ribosomes are able to bind a variety of NTPs, NDPs and NTP analogues, with a preference for ATP. Effective inhibitors of the ribosomal ATPase are ammonium metavanadate and alcaloid emetine. The ATPase activity is present on both ribosomal subunits, which may reflect the existence of two catalytical sites for ATP on the 80 S ribosome. Ribosomal ATPase is stimulated by the occupancy of the A site, in particular with charged tRNA. The ATPase inhibitor adenylylimidodiphosphate almost completely prevents elongation-factor(EF)-1-dependent binding of Phe-tRNA(Phe) to the A site. The hydrolysis of ATP, therefore, is likely to be involved in the mechanism of tRNA binding to the A site of the 80 S ribosome. As far as wide substrate specificity and possible participation in tRNA interaction with the ribosome are concerned, the ribosomal ATPase seems to be similar to EF-3 found in fungi. A synergism in ATPase activities of yeast EF-3 and rabbit liver ribosomes at high ATP concentration and certain ribosome/EF-3 ratios have been observed. Rabbit liver ribosomes seem to stimulate the ATPase activity of yeast EF-3 similar to the mechanism in yeast ribosomes, though less efficiently.

Fetal fibroblast contraction of collagen matrices in vitro: the effects of epidermal growth factor and transforming growth factor-beta.

Wound contraction is an important component of healing but, in the extreme, may lead to excessive scar formation and pathological wound contracture. Fetal rabbit wounds heal without contraction or scarring, whereas excisional fetal sheep wounds have been shown to contract, but no scarring or pathological wound contracture is noted. We used an in vitro model, the fibroblast-populated collagen lattice, to study the ability of fetal fibroblasts to coordinate contraction of a collagen matrix and the modulating effects of epidermal growth factor and transforming growth factor-beta 1 on this contraction. With increasing gestational age, fibroblasts increased the degree of collagen lattice contraction. Epidermal growth factor inhibited contraction by fetal fibroblasts, whereas transforming growth factor-beta 1 stimulated it. These findings suggest that while intrinsic differences between fetal and adult fibroblasts exist, polypeptide growth factors may operate at the site of tissue repair to alter cell phenotype. Further work is underway to delineate the role of soluble protein factors responsible for the absence of scarring and contracture seen in the fetal wound.

Template-dependent peptide formation on ribosomes catalyzed by pyridine.

We found that poly(U)-dependent polyphenylalanine synthesis on Escherichia coli ribosomes was extremely accelerated in the presence of a high concentration of pyridine. In this reaction, chemical energy sources, such as ATP and GTP, and soluble protein factors are not required, but the template and ribosomes are essential for the progress of the reaction. The reaction was inhibited by the antibiotics which inhibit the usual bacterial translation process on the ribosomes. These observations clearly demonstrate that the pyridine-catalyzed amino acid condensation reaction proceeds on the ribosome.

Chromosomal localization of the IL-6 receptor signal transducing subunit, gp130 (IL6ST)

Signal transduction in eukaryotic cells is a complex process mediated, normally, by the interaction of soluble extrinsic protein factors and their cognate receptors. One example of this phenomena is the inflammatory cytokine interleukin-6 and the IL-6 receptor. However, the IL-6 receptor, once its ligand is bound, associates with another membrane glycoprotein, gp130, to potentiate the cytokine response. To further understand the basis of this interaction, and its possible implications in cellular transforming events, the corresponding gene(s) must be studied. Here we find that the human gp130 gene product is homologous to two distinct chromosomal loci on chromosomes 5 and 17. Furthermore, the presence of two distinct gp130 gene sequences is restricted to primates and is not found in other vertebrates.

No Detection of Characteristic Fungal Protein Elongation Factor EF-3 in Pneumocystis carinii

The taxonomic status of Pneumocystis carinii is uncertain, and P. carinii has been categorized both as a fungus and as a protozoan. Recent comparisons of RNA sequence homologies between P. carinii and several genera of fungi and protozoa suggest that P. carinii has closer affinities with the ascomycetes than with the protozoa. The translatory systems of the fungi, however, require three soluble protein factors for peptide chain elongation rather than the two necessary in other eukaryotic systems; to date the additional protein elongation factor (EF-3) appears to be unique to fungi. Western blot analysis of cell-free extracts of P. carinii, derived from rat, was done using a polyclonal antibody raised in rabbits to Saccharomyces cerevisiae EF-3. Anti-EF-3 cross-reacting material was detected only in lysates of Candida albicans and S. cerevisiae included as fungal controls; no cross reaction was detected in lysates of P. carinii, P. carinii-infected rat lung, or a protozoan control (Trichomonas vaginalis).

Structure–function relationships in eukaryotic nuclei

It may be that eukaryotic nuclei contain a collection of operationally independent units (genes), each controlled through its interactions with soluble protein factors which diffuse at random throughout the nucleoplasmic space. Alternatively, nuclei might be organized in such a sophisticated fashion that specific genes occupy distinct sites and that spatially ordered RNA synthesis, processing and transport delivers mature RNAs to predestined sites in the cytoplasm. Different fields of research support each of these extreme views. Molecular biologists inspecting the precise details of specific interactions, usually in vitro, inevitably favour the former, while cell biologists working with far more complicated systems generally assume that more elaborate arrangements exist. In considering the importance of nuclear architecture, I have attempted to relate a collection of experiments each of which intimates some close relationship between structural aspects of chromatin organization and the precise mechanisms underlying nuclear function. I will argue that higher-order structures are crucial for achieving the observed efficiency and coordination of many nuclear processes.

Effect of spermine on transfer RNA and transfer RNA-ribosome interactions.

Translation of genetic message requires a coordianted interplay of more than a hundred kinds of macromolecules. Besides the ribosomes, which are multicomponent ribonucleoprotein particles by their own nature, molecules of transfer RNA, aminoacyl-tRNA synthetases, soluble protein factors and mRNA are involved in the process. Ribosomes provide an unspecific stage for the codon-anticodon interaction and catalyze the peptide bond formation. Molecules of tRNA play a crucial and highly specific role in the over-all process: they are recognized by specific aminoacyl-tRNA synthetases to be charged with their cognate amino acids; the resulting aminoacyl-tRNAs are then brought, in the form of ternary complexes with GTP and the elongation factor Tu, to the decoding or A site of the ribo-some. There they are screened for the proper codon-anticodon matching and only the correct aminoacyl-tRNA is allowed to enter the transpeptidation reaction. The resulting peptidyl-tRNA is then translocated to the peptidyl or P site to serve as the peptidyl donor in the next round of translation. The process in its simplified form is shown in Figure 1.

The function, structure and regulation of E. coli peptide chain release factors

The termination of protein synthesis in Escherichia coli depends upon the soluble protein factors RF1 or RF2. RF1 catalyzes UAG and UAA dependent termination, while RF2 catalyzes UGA and UAA dependent termination. The proteins have been purified to homogeneity, their respective genes isolated, and their primary structures deduced from the DNA sequences. The sequences reveal considerable conserved homology, presumably reflecting functional similarities and a common ancestral origin. The RFs are encoded as single copy genes on the bacterial chromosome. RF2 exhibits autogenous regulation in an in vitro translation system. The mechanism of autoregulation appears to be an in-frame UGA stop codon that requires a 1 + frameshift for the continued synthesis of the protein. Frameshifting prior to the inframe stop codon occurs at a remarkably high frequency by an unknown mechanism. Future studies will be directed at understanding how RFs interact with the ribosomal components, and further defining the mechanism of RF2 frameshifting.

Isolation of an MSP1-derived transcriptionally active nucleolar particle

A discrete macromolecular structure with inherent transcription activity has been isolated from Novikoff nucleoli. The particle (350 Å diameter) was released from the intact nucleoli by digestion with restriction endonuclease (Msp1) and is enriched in repetitive DNA sequences both 5′ and 3′ to the ribosomal genes. In vitro transcription analyses suggest that the particle contains the rDNA polymerase template, and that its 12–15 major proteins are responsible for transcriptional activity without added soluble protein factors.

27] Soluble protein factors and ribosomal subunits from yeast. Interactions with aminoacyl-tRNA

The elongation of the peptide chain on the ribosomes requires the participation of protein factors which are present in the soluble fraction of both prokaryotic and eukaryotic ceils. Yeast has two different protein synthesizing systems: a mammalian-type one in the cytoplasm 1 and other in the mitochondria with properties similar to those from bacteria. This chapter describes the preparation of a supernatant fraction from the yeast Saccharomyces ragilis × Saccharomyces dobzanskff and the interactions of this fraction with aminoacyl and N-acetylaminoacyl-tRNA, the isolation from this supernatant fraction of partially purified elongation factors, and the preparation of ribosomes and active ribosomal subunits and their interactions with aminoacyl-tRNA.

The role of soluble protein factors in the translational control of protein synthesis in eukaryotic cells

The role of soluble protein factors in the translational control of protein synthesis in eukaryotic cells

Atypical Transfer RNA's And Their Origin In Neoplastic Cells

Publisher Summary This chapter focuses on the structure and synthesis of transfer RNA (tRNA) and discusses the biochemistry of tRNA methylases. Translation is the most complex molecular mechanism known in the living cell. It requires messenger RNA, which is the simplest of the structures involved, even though it is laden with information. The ribosomes are complex structures composed of RNA and at least a score of different proteins. Transfer RNA is the most complex biomacromolecule known. In addition to these major components, there are ancillary ones— enzymes that transfer amino acids to the transfer RNA. Moreover, there are at least 8 soluble protein factors needed for the completion of the synthesis of a protein. Any or all of these numerous components could be a regulatory factor in protein synthesis. There are three different lines of biological evidence that point to transfer RNA as a regulatory factor. Transfer RNA is a pivotal molecule in protein synthesis. It is the link between the amino acids and the message-bearing nucleic acids. The tRNA methylases are a complex family of enzymes that modify the structure of preformed tRNA by the insertion of methyl groups into specific positions in the four main bases of tRNA. The enzymes are species-specific, organ-specific, base-specific, and even site-specific for particular bases.

Interaction of transferase II with the 60 s ribosomal subunit

Elongation of polypeptide chains in mammalian systems requires two soluble protein factors, transferases I and II. Transferase I has been shown to function in the binding of aminoacyl-tRNA to ribosomes [ 1,2] , and evidence has been reported that transferase I (or a complex containing it) interacts specifically with the 40 S ribosomal subunit [3]. Transferase II has been demonstrated to function in translocation of peptidyl-tRNA from the acceptor to the donor site on ribosomes [2, 4,5] , but the exact location of its action is not known. Here we report evidence that transferase II from rabbit reticulocytes binds specifically to the 60 S ribosomal subunit. These results correlate with reports of a specific interaction of the translocation factor from E. coli (G factor) with the larger (50 S) ribosomal subunit from that organism [6,7] . Data are also presented on the distribution of transferase II in the supernatant and ribosomal fraction of reticulocytes.

Protein chain initiation by methionyl-tRNA in wheat embryo.

The mechanism of protein chain initiation has been investigated in a cell-free amino acid incorporation system from wheat embryos dependent on tobacco mosaic virus RNA. Analysis of the N-termini of the labeled peptide products of short-term incubations showed the presence of unblocked methionine. In addition, methionyl-tRNA (Met-tRNA) could be bound to ribosomes at 1.3 mM Mg(++) in a reaction requiring viral RNA, ATP, GTP, and soluble protein factors. Incorporation experiments with the two cytoplasmic Met-tRNAs of wheat germ, an initiating species designated Met-tRNA(i) and a Met-tRNA(m), showed that methionine transfer from Met-tRNA(i) was linear from zero time, while that from Met-tRNA(m) occurred only after an appreciable lag. Analysis of the peptide products showed that methionine transfer from Met-tRNA(i) was predominantly N-terminal. In contrast, methionine transfer from Met-tRNA(m) was exclusively into internal positions. Similar selectivity was observed in the ribosome binding assay; only Met-tRNA(i) showed a strong reaction. These experiments provide strong evidence that in the wheat embryo, cytoplasmic Met-tRNA(i) functions without formylation in the initiation of protein synthesis.

Purification of Aminoacyltransferase II (Translocation Factor) from Rat Liver

Abstract Aminoacyltransferase II is one of the two soluble protein factors required for polypeptide biosynthesis from aminoacyl transfer RNA with rat liver preparations. This transfer factor and guanosine triphosphate are required for the translocation of messenger RNA and peptidyl transfer RNA from the aminoacyl to the peptidyl site on ribosomes. A method for the purification of the translocation factor has been devised. After the resolution of the translocase from the other transfer factor (aminoacyltransferase I) on hydroxylapatite, the translocase is chromatographed on cellulose phosphate and DEAESephadex columns and then subjected to electrophoresis in an electrofocusing (sucrose density-pH gradient) apparatus. The final product seems to be homogenous on acrylamide gel electrophoresis and ultracentrifugation. A molecular weight estimate of 60,000 to 65,000 has been made. Specific activities, up to 1,000-fold higher than that of the pH 5 supernatant fraction of rat liver homogenates, the starting material for these studies, have been obtained.

臓器組織と鉛との結合状態に関する実験的研究 : Ca-EDTAの応用

By a previous author in our department, an experimental study had been made on the state of combination of lead in blood, applying the chelating action of Edathamil. In this study, the present author performed a similar experiment on organ-homogenates such as of liver, brain, lung, muscle, etc., in order to make overt the difference of combining affinity with lead by variable organs. The following conclusions were reached by the experiment. 1) In the liver of normal rabbits, there is such relation between the dose of added lead and the composition of Pb-EDTA as is expressed by an equation: y=4.70x0.84, where y=dose of added, x=lead amount of Pb-EDTA. 2) The added lead into organ-homogenates of normal rabbits in vitro is combined with bone and liver more firmly than with brain or lung. 3) Lead in organs of rabbits with chronic lead poisoning is more stable to be combined, compared with lead in those with acute lead poisoning. 4) The different state of lead combination with variable organs or the difference of affinity with lead by organ is not only attributed to the different chemical property of water non-soluble protein, but might also be due to water soluble protein and other biological factors which are not detected by the present experiment.