Aminoacyl Transfer Ribonucleic Acid Research Articles

The Effect of Amino Acid Analogues on the Aminoacylation of Transfer Ribonucleic Acid

The Effect of Amino Acid Analogues on the Aminoacylation of Transfer Ribonucleic Acid

Mescaline-induced changes of brain cortex ribosomes: Effect of mescaline on the binding of aminoacyl-transfer ribonucleic acid to ribosomes of brain tissue

The effect of mescaline sulfate on the binding of aminoacyl-tRNA by brain cortex ribosomes was studied. The poly U-directed binding of 14C-phenylalanyl-tRNA in vitro was moderately inhibited by mescaline. The pretreatment of normal ribosomes with mescaline caused a decrease of their phenylalanyl-tRNA binding capacity. The pretreatment of brain cortex slices with mescaline also decreased the phenylalanyl-tRNA binding capacity of the ribosomes isolated from the slices so treated. This was true in assays with phenylalanyl-tRNAs from both Escherichia coli and yeast. This inhibitory effect was dependent on the concentration and length of treatment of brain cortex slices with mesaline.

Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver.

Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver.

Pseudoverification of Mixed Aminoacyl Transfer Ribonucleic Acids: THE GENERALITY OF THE PROCESS

Abstract A mixture of unresolved tRNAs from Escherichia coli B has been acylated with a mixture of 18 3H-amino acids, and the purified, correctly acylated aminoacyl-tRNAs were incubated with pure isoleucyl-tRNA synthetase under conditions in which it hydrolyzes some noncognate aminoacyl-tRNAs (see the preceding paper). A micromethod was used to resolve and identify the 3H-amino acids released. The results indicate that isoleucyl-tRNA synthetase can interact with and deacylate 15 of the 18 aminoacyl-tRNA species used. The occurrence of this reaction indicates an interaction between these tRNAs and isoleucyl-tRNA synthetase. In addition, the specificity of the verification, or hydrolysis, reaction is illuminated by the particular nature of the residues which are resistant to hydrolysis.

Mechanism of an aminoacyl transfer ribonucleic acid synthetase

Mechanism of an aminoacyl transfer ribonucleic acid synthetase

Purification of chicken liver seryl transfer ribonucleic acid by complex formation with elongation factor EF-Tu:GTP. A general micromethod of aminoacyl transfer ribonucleic acid purification.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPurification of chicken liver seryl transfer ribonucleic acid by complex formation with elongation factor EF-Tu:GTP. General micromethod of aminoacyl transfer ribonucleic acid purificationBarry J. Klyde and Merton R. BernfieldCite this: Biochemistry 1973, 12, 19, 3752–3757Publication Date (Print):September 1, 1973Publication History Published online1 May 2002Published inissue 1 September 1973https://doi.org/10.1021/bi00743a026RIGHTS & PERMISSIONSArticle Views22Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (648 KB) Get e-Alerts Get e-Alerts

Mode of action of lividomycin on protein synthesis in Escherichia coli.

Lividomycin specifically inhibited bacterial protein synthesis and had codon misreading activity. Lividomycin, moreover, stimulated the binding of aminoacyl-transfer ribonucleic acid to ribosomes, but did not show any significant effects on the formation of aminoacyl-transfer ribonucleic acid and the puromycin reaction.

Effect of puromycin analogues and other agents on peptidyl-puromycin synthesis on polyribosomes.

The incorporation of [(3)H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. The K(m) for puromycin is 4 muM with bacterial polyribosomes; under the same conditions, the K(i) for psi-hydroxy-puromycin (6-dimethylamino-9-[3-(l-beta-phenyllactylamino)-3-deoxy-beta- d-ribofuranosyl] purine) is 240 muM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl-l-alanylamino)]-cyclopentyl}purine) is 1 muM. Both were found to be competitive inhibitors of puromycin. The K(m) for C-A-C-C-A(Phe) is 250 muM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 muM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the alpha-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5'-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively high K(m) for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.

Interaction of rabbit reticulocyte elongation factor 1 with guanosine-triphosphate and aminoacyl-transfer ribonucleic acid

Elongation Factor 1 (EF-1) from rabbit reticulocytes interacts with GTP to form a complex that is retained on a nitrocellulose filter. EF-1 also interacts with GDP; however, the concentration of GDP required for maximal complex formation is higher than the concentration of GTP required and the extent of binding is lower. Interaction of EF-1 with GTP in the presence of various aminoacyl-tRNAs from rabbit liver or E. coli results in a 50–75% decrease in the amount of GTP complex retained on a filter. No reduction in the amount of GTP complex retained is observed with deacylated tRNA or with N-acetylphenylalanyl-tRNA. EF-1 is inactivated by heating at 37 °C in the presence of GTP. Aminoacyl-tRNA protects EF-1 from the inactivation observed in the presence of GTP. These data indicate that an interaction of reticulocyte EF-1 with GTP and aminoacyl-tRNA occurs; however, attempts to demonstrate the formation of a stable ternary complex by chromatography on Sephadex G-150 were unsuccessful. Also, no difference is observed between the rate of binding of aminoacyl-tRNA to reticulocyte ribosomes obtained with EF-1 and the rate obtained with EF-1 that had been incubated previously with GTP and aminoacyltRNA.

A temperature-sensitive glycyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

A method of selecting temperature-sensitive mutants of Escherichia coli which makes use of a double temperature shift combined with suicide as a result of incorporated 3H-uridine is described. One mutant selected in this way shows restricted growth at 42 °C resulting from early inhibition of protein and ribonucleic acid (RNA) synthesis. In vitro assays of aminoacyl-transfer RNA synthetase activity indicated a complete absence of active glycyl-transfer RNA synthetase in the mutant. This enzyme activity in the mutant was distinguishable from the wild type by its rapid inactivation at 28 °C in cell-free extracts. Infection of the temperature-sensitive mutant with bacteriophage T4 did not alter the heat-sensitivity of the mutant enzyme. The mutation is 72% cotransducible with the xyl locus.

Aminoacylation of transfer ribonucleic acid in vitro during the mitotic cycle of Physarum polycephalum.

Aminoacylation of transfer ribonucleic acid in vitro during the mitotic cycle of Physarum polycephalum.

Isolation and characterization of a regulatory mutant of an aminoacyl-transfer ribonucleic acid synthetase in Escherichia coli K-12.

From Escherichia coli strain K28, which is temperature sensitive for growth because of a mutation in its seryl-transfer ribonucleic acid (tRNA) synthetase gene (serS), temperature-resistant mutants were selected which were found to have a fivefold higher level of seryl-tRNA synthetase than the parent strain. The "high-level" character was found to be genetically stable and is due to a mutation in a locus denoted serO. This locus was found to be very closely linked to serS on the genetic map, and the relative gene order was concluded to be serS-serO-serC. In a serO(-) strain, the normal dependence of seryl-tRNA synthetase (SerRS) activity on changes of exogenous serine concentration was not observed. In a stable heterozygous merodiploid, the serO(-) mutation is still expressed, i.e., it is cis dominant. These results strongly suggest that serO is an operator site involved in the control of the serS gene.

On the conservation of aminoacyl-transfer ribonucleic acid synthetase genes in Bacillus.

None of the heterologous deoxyribonucleic acid from various bacilli was capable of transforming lysyl- and tryptophanyl-transfer ribonucleic acid (tRNA) synthetase mutants of Bacillus subtilis to wild type. It was concluded that there is little conservation of the aminoacyl-tRNA synthetases even though the tRNA cistrons are conserved genetic functions.

Changes in certain aminoacyl transfer ribonucleic Acid synthetase activities in developing pea roots.

Tyrosyl-, arginyl-, leucyl-, and phenylalanyl-tRNA synthetase activities were measured in extracts from three root sections of 3-day-old pea seedlings. The sections 0 to 2, 3 to 7, and 8 to 22 millimeters from the root tip were chosen to represent the regions of cell division, elongation, and maturation, respectively. The specific activity for each aminoacyl-tRNA synthetase was highest in the 0- to 2-millimeter section and lowest in the 8 to 22 millimeter section. The changes in specific activity between the sections, however, varied with the particular synthetase. Tyrosyl-tRNA synthetase from each section was fractionated into two activity regions on a diethylaminoethyl cellulose column. Approximately 10, 22, and 44% of the total tyrosyl-tRNA synthetase activity in the 0 to 2, 3 to 7, and 8- to 22-millimeter sections, respectively, were associated with the first tyrosyl-tRNA synthetase region; the remaining activity was located in the second tyrosyl-tRNA synthetase region. Only one activity region for arginyl-tRNA synthetase was detected by diethylaminoethyl cellulose column fractionation.

Release of aminoacyl transfer ribonucleic acid from transfer ribonucleic acid synthetase studied by rapid molecular sieve chromatography

Complex of Ile-tRNA Ile and isoleucyl-tRNA synthetase (IRS) was isolated by rapid chromatography on a Bio-Gel P-100 column at 4°C. By incubating the complex in the presence of excess unacylated tRNA Ile prior to chromatography, it is possible to qualitatively measure the rate of exchange of Ile-tRNA Ile with tRNA Ile on the enzyme. The rate of exchange is markedly accelerated by isoleucine and isoleucinyl-AMP, but not by ATP. These results confirm previously published findings that the rate of release of newly synthesized Ile-tRNA Ile from IRS is very slow in the absence of isoleucine or isoleucyl-AMP, but that the release is greatly enhanced by these ligands. The rapid chromatography procedure thus provides a very direct and straightforward means for measuring the dynamics of a proteinnucleic acid interaction.

Inhibition by Aminoacyl Transfer Ribonucleic Acid of Elongation Factor G-dependent Binding of Guanosine Nucleotide to Ribosomes

Abstract Ribosomes complexed with poly(U) and Phe-transfer RNA (tRNA) have been examined for their ability to interact with elongation factor G (EF-G). It is shown that Phe-tRNA, bound to ribosomes either at 6 mm Mg2+ in the presence of elongation factor Tu and GTP or at 20 mm Mg2+ in the absence of these compounds, strongly inhibits (50 to 90%) the binding of EF-G, as measured by the formation of either the GDP·EF-G·fusidic acid·ribosome complex or the 5'-guanylyl methylene diphosphonate·EF-G·ribosome complex. Moreover, ribosome-bound Phe-tRNA inhibits also, and to a similar extent, both the GTP hydrolysis associated with the formation of the GDP·EF-G·fusidic acid·ribosome complex and that observed in the presence of only EF-G and ribosomes. These findings, together with the previously observed inhibition of aminoacyl-tRNA binding by ribosome-bound EF-G, lead to the conclusion that EF-G and aminoacyl-tRNA bind to the ribosome at mutually exclusive sites. This provides strong evidence for overlapping or interaction between the binding site of EF-G on the 50 S subunit and that of aminoacyl-tRNA, the ribosomal A-site.

The Role of Guanosine Triphosphate Hydrolysis in Elongation Factor Tu-promoted Binding of Aminoacyl Transfer Ribonucleic Acid to Ribosomes

Abstract The role of guanosine triphosphate in the polypeptide elongation factor Tu (EF-Tu)-promoted binding of aminoacyl-tRNA to ribosomes has been investigated. In the presence of EF-Tu and GTP, the binding of phenylalanyl-tRNA to the A site of the poly (U)-ribosome complex having an N-acetylphenylalanyl-tRNA prebound to its P site (Complex I) resulted in the formation of N-acetyldiphenylalanyl-tRNA. 5'-Guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP, could substitute for GTP in promoting the binding of phenylalanyl-tRNA to ribosomes in the presence of stoichiometric amount of EF-Tu. However, very little dipeptide formation was observed under this condition. In contrast, when the complex formed with Gpp(CH2)p was incubated at 37° for 5 min and reisolated by centrifugation through 10% sucrose, approximately 50% of the bound radioactivity was recovered as N-acetyldiphenylalanine. This is presumably due to the detachment of Tu-Gpp(CH2)p from ribosomes during reisolation. It was concluded that the role of GTP hydrolysis in this reaction is to remove EF-Tu from ribosomes after the completion of the binding of aminoacyl-tRNA. The release of EF-Tu may be necessary to facilitate the peptidyl transfer and also to initiate the cyclic reutilization of EF-Tu.

Inhibition of cell-free protein synthesis by hydrostatic pressure.

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.

Methionine Transfer Ribonucleic Acid from Folate-sufficient and Folate-deficient Streptococcus faecalis R

Abstract The formation of aminoacyl transfer ribonucleic acid derivatives containing a blocked α-amino group such as the formylmethionyl ester of methionine transfer ribonucleic acid (fMet-tRNAfmet) was studied in Streptococcus faecalis R. This bacterium cannot synthesize folate, and its growth is dependent upon the presence of either added folic acid or a mixture of products of biosynthetic reactions requiring the participation of tetrahydrofolate: serine, methionine, a purine, and thymine. Only extracts prepared from S. faecalis R cells grown on medium containing folic acid catalyzed the formation of an amino acid derivative esterified to tRNA which lacked a free α-amino group; of the amino acids tested, only methionine was a substrate for the formation of such an aminoacyl-tRNA derivative containing a blocked α-amino group. The methionyl-tRNA derivative was identified as N-formylmethionyl-tRNA. Extracts prepared from S. faecalis R cells grown on folate-free medium supplemented with serine, methionine, a purine, and thymine could not catalyze the formation of any detectable aminoacyl-tRNA species containing a modified α-amino group. The in vivo level of charged tRNAmet of exponentially grown S. faecalis R was estimated to be near 100% for both folate-sufficient and folate-deficient grown cells, and the total amount of tRNAfmet was the same under both culture conditions. However, methionyl-tRNAfmet was only formylated in cells grown in the presence of folic acid. Approximately 82 to 85% of the tRNAfmet of S. faecalis R in exponential growth stage in folate-containing medium was blocked to further formylation using 10-[14C]formyltetrahydrofolate as the formyl donor, whereas none of the tRNAfmet prepared from cells grown in the absence of folic acid was blocked to formylation. These results indicate that methionyl-tRNA in S. faecalis R grown in the absence of folic acid but in the presence of the supplements is neither formylated nor contains a blocked α-amino group and provide further evidence that formylation of methionyl-tRNAfmet is not required for the initiation of protein synthesis in S. faecalis R under certain defined conditions of culture.

Temperature-sensitive osmotic remedial mutants of Escherichia coli.

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.