Participation In Protein Synthesis Research Articles

Physiological roles of tryptophan in teleosts: current knowledge and perspectives for future studies

AbstractTryptophan is an essential amino acid with a huge functional versatility, in addition to its participation in protein synthesis. Because of the complexity of its metabolism, and the functional relevance of several of its metabolites, it directly or indirectly participates in a wide array of physiological pathways. This amino acid is a precursor for the synthesis of the neurotransmitter/neuromodulator serotonin (5HT), the hormone melatonin and kynurenine and related compounds such as kynurenic acid, quinolinic acid or niacin. Because of this, it has a key role in the regulation of processes ranging from the neuroendocrine to the immune system in vertebrates. In aquaculture, extensive research has been performed to optimize the levels of tryptophan in the commercial diets for many fish species. Providing adequate levels of this amino acid is critically important for fish growth but also for fish welfare, as tryptophan has been shown to modulate fish behaviour, stress responses, and antioxidant and immune systems. Currently, available data suggest a wide variation in tryptophan requirements of different species ranging 0.3–1.3% of dietary protein level, but recent evidence also shows that fish tryptophan requirements can greatly vary depending on the rearing conditions of the fish. We also review here the participation of tryptophan and related metabolites in different physiological functions that are crucial for fish welfare. The review covers the involvement of tryptophan in 5HT‐ and melatonin‐mediated functions, along with its participation in the regulation of the immune system and its role as an antioxidant and antitoxic agent in fish.

Structural Basis for the Exceptional Stability of Bisaminoacylated Nucleotides and Transfer RNAs

At least one bisaminoacyl-tRNA is synthesized in nature (by Thermus thermophilus phenylalanyl-tRNA synthetase), and many disubstituted tRNAs have been prepared in vitro. Such misacylated tRNAs are able to participate in protein synthesis, even though they lack the free 2'-OH group of the 3'-terminal adenosine moiety. Their ready participation in protein synthesis implies significant chemical reactivity. The basis for this reactivity has been documented previously. Surprisingly, the aminoacyl moieties of these tRNAs also exhibit exceptional chemical stability. In the present report, bisaminoacylated nucleotides are investigated computationally and experimentally to define the basis for the stability of such species. Molecular modeling of bisalanyl-AMP in the absence of solvent and in the presence of a limited number of water molecules revealed two common features among the low-energy structures. The first was the presence of H-bonding interactions between the two aminoacyl moieties. The second was the presence of a H-bonding interaction between the 2'-O-alanyl moiety and the N-3 atom of the adenine nucleobase, typically mediated through a water molecule. The prediction of an interaction between an aminoacyl moiety and the adenine nucleobase was confirmed experimentally by comparing the behavior of bisalanyl-AMP and bisalanyl-UMP in the presence of model nucleophiles. This study suggests a possible role for the adenosine moiety at the 3'-end of aminoanyl-tRNAs in controlling the stability and reactivity of the aminoacyl moiety and has important implications for the reactivity and stability of normal aminoacyl-tRNAs.

Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

DNA repair efficiency in transgenic mice over expressing ribosomal protein S3

Human ribosomal protein S3 (RPS3) has previously been shown to have alternative roles beyond its participation in protein synthesis. For example, our in vitro studies have shown that RPS3 has an extraordinarily high binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG). Notably, in cells exposed to oxidative stress RPS3 translocates to the nucleus where it co-localizes with foci of 8-oxoG. We have engineered transgenic mice over expressing RPS3 in an attempt to determine the outcome of RPS3 translocation in a whole animal. Mouse embryonic fibroblasts (MEFs) isolated from these transgenic mice showed an increased accumulation of DNA damage in cells exposed to oxidative damage when compared to MEFs from wild-type mice. In MEFs exposed to oxidative stress we observed the translocation of RPS3 from the cytoplasm to the nucleus and co-localizing to 8-oxoG foci, an observation that could involve the blocking of the repair of this mutagenic base and thereby explain why transgenic MEFs exposed to oxidative stress have higher levels of DNA damage.

Insights into functional modulation of catalytic RNA activity

RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

Modeling the reactive properties of tandemly activated tRNAs

Tandemly activated tRNAs, bearing amino acid moieties at both the 2'- and 3'-positions of the 3'-terminal adenosine moiety (A(76)), have been shown to participate efficiently in protein synthesis [B. Wang, J. Zhou, M. Lodder, R. D. Anderson, III and S. M. Hecht, J. Biol. Chem., 2006, 281, 13865]. The mechanism by which such activated tRNAs are able to donate both amino acids to the growing polypeptide chain is not well understood. Here we report the chemical behavior and participation in protein synthesis of new bisaminoacyl derivatives of pdCpA and tRNA. Both amino moieties of the aminoacyl groups are shown to be important to enable participation in protein synthesis; paradoxically, they also confer an unanticipated chemical stability toward different nucleophiles. The results obtained suggest a model for participation of bisaminoacylated tRNAs in protein synthesis.

Synthesis of bisaminoacylated pdCpAs and tandemly activated transfer RNAs

Described herein is the preparation of new bisacylated tRNAs and their participation in protein synthesis. It has been reported that Thermus thermophilus phenylalanyl-tRNA synthetase can introduce two phenylalanine moieties onto the 3′-terminal adenosine of its cognate tRNA. It is also possible to prepare bisactivated tRNAs in vitro; these participate in protein synthesis [Wang, B.; Zhou, J.; Lodder, M.; Anderson, R. D.; Hecht, S. M. J. Biol. Chem. 2006, 281, 13865]. Presently, the chemical strategy used for the synthesis of the key intermediate bisacylated pdCpAs is described. Bis- S-alanyl- and bis- S-methionyl-pdCpAs were prepared initially. Further, S-threonine, S- allo-threonine, S-homoserine, and ( S)-(+)-2-amino-3-hydroxy-3-methylbutyric acid were coupled with the dinucleotide to define preparative methods applicable to more complex amino acids bearing additional functionality in the form of an OH group.

RIBOSOME-INACTIVATING PROTEINS: A Plant Perspective.

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved alpha-sarcin loop of large rRNAs. This depurination inactivates the ribosome, thereby blocking its further participation in protein synthesis. RIPs are widely distributed among different plant genera and within a variety of different tissues. Recent work has shown that enzymatic activity of at least some RIPs is not limited to site-specific action on the large rRNAs of ribosomes but extends to depurination and even nucleic acid scission of other targets. Characterization of the physiological effects of RIPs on mammalian cells has implicated apoptotic pathways. For plants, RIPs have been linked to defense by antiviral, antifungal, and insecticidal properties demonstrated in vitro and in transgenic plants. How these effects are brought about, however, remains unresolved. At the least, these results, together with others summarized here, point to a complex biological role. With genetic, genomic, molecular, and structural tools now available for integrating different experimental approaches, we should further our understanding of these multifunctional proteins and their physiological functions in plants.

Participation of Protein Synthesis in in vitro Oocyte Maturation and Fertilization in Cattle

Participation of Protein Synthesis in in vitro Oocyte Maturation and Fertilization in Cattle

Growth hormone release from chicken anterior pituitary cells in primary culture: TRH and hpGRF synergy, protein synthesis, and cyclic adenosine 3′5′-monophosphate

Our earlier work showed that the effects of thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) on growth hormone (GH) release are synergistic (greater than additive) in a primary culture of chicken adenohypophyseal cells. The purpose of the present studies was to investigate the possible participation of protein synthesis and cyclic adenosine 3′5′-monophosphate (cAMP) in GH release. Following culture (48 hr), cells were incubated for 2 hr with test agents. Cycloheximide (an inhibitor of protein synthesis) had no effect on basal (absence of test agent) GH release or hpGRF-induced GH release. However, cycloheximide abolished the synergy between TRH and hpGRF. Although neither TRH nor hpGRF alone stimulated GH production (intracellular GH plus GH release) during a 2-hr incubation period, in combination these secretagogues increased total GH. These findings suggest that GH release from the chicken somatotroph under conditions of TRH and hpGRF synergy requires protein synthesis. In other studies, cells were exposed to agents inducing the formation of cAMP and either TRH or hpGRF. 8 Br-cAMP (10 −3 M), forskolin (10 −6 M), or isobutylmethylxanthine (IBMX; 10 −3 M) alone stimulated GH release to values between 30 and 50% over the basal value. The combined effects of each of these agents and TRH on GH release were synergistic. Similarly, IBMX and hpGRF exerted synergistic effects on GH release. In contrast, no synergy was shown between hpGRF and either 8 Br-cAMP or forskolin; their combined actions were less than additive.

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis.

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.

Participation of Protein Synthesis in Development of Supersensitivity in Cultured Rat Vas Deferens

Organ culture of rat vas deferens produced supersensitivity to norepinephrine and acetylcholine in contractile response without change in α1-adrenergic and muscarinic acetylcholine receptors. The development of supersensitivity was inhibited by low temperature and protein synthesis inhibitors. However, protein synthesis inhibitor had no significant effect on the receptors. These findings suggested that the supersensitivity may be have a stimulatory effect in a process(es) after activation of receptor to contraction.

The Inefficiency of Ribosomes Functioning in Escherichia coli Growing at Moderate Rates

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.

Dissociation of single ribosomes as a preliminary step for their participation in protein synthesis.

32P‐labelled derived 40‐S ribosomal subunits and 80‐S monoribosomes entered the polyribosome fraction of a rabbit‐reticulocyte lysate active in protein synthesis by a process that was dependent on time, energy and temperature. Both 40‐S and 60‐S subparticles from the labelled monoribosome entered the polyribosomes at the same rate. The addition of excess unlabelled 40‐S subparticles to the cell‐free system containing labelled monoribosomes increased the ratio of 60‐S to 40‐S‐subparticle radioactivity in the polyribosome fraction. This indicated a competition between added unlabelled and monoribosome‐derived 40‐S subparticles for entry into polyribosomes. From the evaluation of the translation time of mRNA, of the rate of monoribosome‐polyribosome exchange, and of the equilibrium distribution of the cycling labelled monoribosomes and unlabelled subparticles with polyribosomes it is concluded that the dissociation of single ribosomes into subunits is an obligatory step for their entry into the polyribosoma1 fraction and that this process is coupled with initiation of mRNA translation.

Accumulation of 70S monoribosomes in Escherichia coli after energy source shift-down.

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.

Reversible disaggregation by NaF of membrane-bound polyribosomes of mouse myeloma cells in tissue culture

Approximately one-quarter of the ribosomes of P-3 mouse myeloma cells in tissue culture are bound to membranes of the endoplasmic reticulum. Incubation with NaF causes polyribosome disaggregation; at the same time approximately 25 % of the membrane-bound ribosomes are released from membranes. After reversing the inhibition of NaF free ribosomes become associated with membranes until approximately the same proportion of free and membrane-bound ribosomes found in untreated cells is reestablished. During this time these cells resume the normal synthesis and secretion of immunoglobulin. This takes place even when the synthesis of RNA is completely inhibited. These experiments have suggested that the association of a class of ribosomes with membranes is dependent on their entry into polyribosomes active in protein synthesis whereas attachment of the other ribosomes occurs independently of their participation in protein synthesis.

Regulation of messenger RNA synthesis in Escherichia coli

Quantitative estimates of messenger activity have been obtained using RNA extracted from Escherichia coli to stimulate in vitro protein synthesis. The activity in extracts from cultures grown at different rates was nearly proportional to the amount of ribosomal RNA. In a mutant rich in 43 s ribosomal precursors and 30 s particles the same relationship was found. This suggests that the 30 s particle is active in releasing the messenger RNA from the template and might regulate messenger RNA synthesis. In stringent cells previously depleted of messenger by uracil starvation, messenger RNA synthesis was found to occur at identical rates in the presence or absence of a required amino acid upon re-addition of uracil. These results are compatible with a model in which the synthesis of messenger RNA is regulated by the 30 s particle independent of its participation in protein synthesis.

Biosynthesis of the Peptidoglycan of Bacterial Cell Walls: VII. Incorporation of Serine and Glycine into Interpeptide Bridges in Staphylococcus Epidermidis

Abstract The peptidoglycan of Staphylococcus epidermidis contains serine and glycine in the interpeptide bridges. A particulate enzyme system has been obtained which catalyzes peptidoglycan synthesis from the uridine nucleotides substrates and a transfer RNA (tRNA)-dependent incorporation of both serine and glycine into the peptidoglycan. Maximum serine incorporation requires simultaneous incorporation of glycine, but glycine incorporation is independent of serine incorporation. Serine and glycine are first activated, attached to tRNA, and then transferred to a lipid intermediate of peptidoglycan synthesis. Finally, this lipid intermediate is utilized in the biosynthesis of the peptidoglycan. The seryl-tRNAs of this organism have been separated into four seryl-tRNA fractions. Three of these function both in peptidoglycan synthesis and in protein synthesis, as examined by various methods. The fourth seryl-tRNA participates equally well in peptidoglycan synthesis, but it has not been possible to demonstrate its participation in protein synthesis or in triplet-dependent binding of aminoacyl-tRNA to ribosomes by using triplets corresponding to all of the known serine codons.

Reaction of adenine-specific antibodies with denatured deoxyribonucleic acid.

AT the present time there is no doubt regarding the role of nucleic acids in carrying genetic information, and their participation in protein synthesis and other biological events is now well known1. There is therefore considerable biochemical and genetical importance in the availability of specific antibodies capable of reacting with nucleic acids. The production of such antibodies has been described recently as the result of immunization with DNA that had been complexed with methylated bovine serum albumin2, by coupling periodate-oxidized nucleotides or nucleosides to proteins3, or by binding uridine-5′-carboxylic acid to a synthetic polypeptide4. Such data promise to make the specificity and sensitivity of immunochemical procedures, which have been of great value in the study of other polymers such as polysaccharides and proteins5, similarly available for an analysis of nucleic acids.

Bacterial polyribosomes and their participation in protein synthesis in vivo

In several species of bacteria, polyribosomes are demonstrable in lysates prepared by osmotic lysis of protoplasts or by breakage of whole cells in a French cell. These components have a broad distribution of sedimentation coefficients ranging from 120 s to 400 s in Bacillus megaterium and from 120 s to 300 s in Salmonella typhimurium . They constitute about half of the total ribosomal material in B. megaterium and about one-third in S. typhimurium . They can be partially resolved into individual components by zone centrifugation through density gradients. These structures are converted to 70 s ribosomes by the action of ribonuclease. Labeling of growing cells with amino acids for short times leads to greater incorporation of radioactive material into polyribosomes than into single ribosomes. The subsequent addition of unlabeled amino acids results in faster removal of radioactive material from polyribosomes than from single ribosomes. This is taken as an indication of in vivo involvement of polyribosomes in protein synthesis.