Aminoacyl Transfer Ribonucleic Acid Synthetases Research Articles

Two photo-cross-linked complexes of isoleucine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Escherichia coli Ile-tRNA synthetase and tRNA-Ile have been cross-linked photochemically by the direct action of ultraviolet light. In addition, photo-induced joining of tRNA-Ile E. coli to Val-tRNA synthetase from yeast has also been achieved. This yeast enzyme is known to mischarge E. coli tRNA-Ile with valine. Regions on tRNA-Ile involved in cross-linking have been determined for both complexes. In each case, three distinct parts of the nucleic acid are found cross-linked. Two of these are the same in both complexes and involve the dihydrouridine stem and loop region. The third part is unique for each complex. It involves the 3' terminus in the cognate one and the 3' side of the anticodon in the non-cognate case. When the cross-linked regions are projected onto a model of the three-dimensional structure of tRNA, it is clear that these and other data are consistent with having each enzyme bound in a similar orientation across tRNA-Ile. The enzymes are viewed as spanning the distance from the anticodon to the 3' terminus and making extensive contact with the area in which the two helical branches of the L-shape tRNA structure come together.

The role of polyamines in the aminoacyl transfer ribonucleic acid synthetase reactions. Demonstration of the requirement for magnesium ion and a secondary stimulatory effect of spermine.

Spermine and related polyamines have been reported to substitute for Mg2+ in the aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases, but not in the ATP-PP-i exchange reaction. Such observations have led some workers to propose that these reactions proceed via a concerted mechanism rather than the usual two-step mechanism involving an aminoacyladenylate intermediate. In an attempt to elucidate the mechanism of the spermine effect on acylation and exchange, both reactions were re-examined using isoleucyl-tRNA synthetase from Escherichia coli. In the absence of added Mg2+ untreated tRNA was acylated in the presence of spermine, but tRNA from which Mg2+ had been scrupulously removed was not. ATP-PP-i exchange was not observed when spermine was used in place of Mg2+; however, if tRNA possessing sequestered Mg2+ was added, the exchange reaction was observed. These data suggest that a primary effect of spermine is to displace bound Mg2+ from tRNA in quantities sufficient to promote both the ATP-PP-i exchange and esterification of tRNA. The previously reported stimulatory effects of polyamines on these reactions are believed to be artifacts due to Mg2+ contamination of tRNA. Providing trace levels of Mg2+ are present, spermine exerts a secondary stimulation of the rate of aminoacylation, the mechanism of which is unknown. The results presented refute arguments that these enzymes proceed by a concerted mechansim and support the intermediacy of aminoacyladenylates.

Regulation of branched-chain aminoacyl-transfer ribonucleic acid synthetases in an ilvDAC deletion strain of Escherichia coli K-12.

Valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetase formation was compared in isogenic strains of Escherichia coli K-12 that differed only in that one strain carried a deletion of three genes of the ilv gene cluster, ilvD, -A, and -C. It was found that: (i) the activities of these synthetases in the deletion strain were less than those in the normal strain during growth in minimal medium supplemented with excess isoleucine, valine, and leucine, and (ii) their stability was reduced in the deletion strain during specific branched-chain amino acid limitations. The results of density-labeling experiments suggest that the in vivo stability of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetases requires some product missing in the ilvDAC deletion strain.

Regulation of synthesis of the branched-chain amino acids and cognate aminoacyl-transfer ribonucleic acid synthetases of Escherichia coli: a common regulatory element.

Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli.

Synthesis of branced-chain aminoacyl-transfer ribonucleid acid synthetases in a Salmonella typhimurium mutant with an altered biosynthetic L-threonine deaminase.

The differential rates of synthesis of the three branched-chain aminoacyl-transfer ribonucleic acid synthetases were measured in Salmonella typhimurium LT-2 and a mutant, ilvA504. The mutant produced an l-threonine deaminase with a decreased affinity for its cofactor, pyridoxal-5'-monophosphate. The addition of pyridoxal-5'-monophosphate to cultures of strain ilvA504 growing in excess isoleucine, valine, and leucine resulted in an increased rate of growth and repression of the synthesis of the isoleucine and valine biosynthetic enzymes. No differences in the rate of synthesis of the branched-chain aminoacyl-transfer ribonucleic acid synthetases were observed in cultures of ilvA504 growing with or without added pyridoxal-5'-monophosphate. The differential rates of synthesis of all three enzymes were similar to the rates measured in strain LT-2. These experiments suggest that different forms of the ilvA gene product are involved in the regulation of the branched-chain amino acid biosynthetic enzymes and the branched-chain aminoacyl-transfer ribonucleic acid synthetases.

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of Escherichia coli

Derepression of Synthesis of the Aminoacyl-Transfer Ribonucleic Acid Synthetases for the Branched-Chain Amino Acids of <i>Escherichia coli</i>

Effect of the Inhibition of Protein Synthesis on the Escherichia coli Cell Envelope

The consequences for cell envelope integrity of Escherichia coli K-12 of the inhibition of protein synthesis by a variety of means have been examined. Protein synthesis was blocked by the antibiotics chloramphenicol and streptomycin, by amino acid starvation of an amino acid auxotroph, and by inactivation of temperature-sensitive aminoacyl transfer ribonucleic acid synthetase and ribosomal mutations. Closely similar morphological and physiological effects were found irrespective of the means by which protein synthesis was blocked. Scanning electron microscopy revealed a spectrum of changes after protein inhibition, with granular material derived from cells and spheroplasts commonly seen. Streptomycin caused additional changes manifested in a collapsed appearance of treated cells. Measurements of the release of lipopolysaccharide from the cell surface, alterations in outer membrane penetrability, and lysis of lysozyme-ethylenediaminetetraacetic acid-treated cultures also showed that the various inhibitory treatments all had similar effects on cell envelope properties. The close correspondence between the effects seen with antibiotic-treated cultures and those in which protein synthesis inhibition was achieved by use of mutants indicates that the effects of chloramphenicol and streptomycin on the cell envelope are indirect consequences of ribosomal block, rather than due to multiple sites of action of the antibiotics.

Derepression of synthesis of the aminoacyl-transfer ribonucleic acid synthetases for the branched-chain amino acids of Escherichia coli.

The kinetics of derepression of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined during valine-, isoleucine-, and leucine-limited growth. When valine was limiting growth, valyl-tRNA synthetase formation was maximally derepressed within 5 min, whereas the rates of synthesis of isoleucyl-, and leucyl-tRNA synthetases were unchanged. Isoleucine-restricted growth caused a maximal derepression of isoleucyl-tRNA synthetase formation in 5 min and derepression of valyl-tRNA synthetase formation in 15 min with no effect on leucyl-tRNA synthetase formation. When leucine was limiting growth, leucyl-tRNA synthetase formation was immediately derepressed, whereas valyl- and isoleucyl-tRNA synthetase formation was unaffected by manipulation of the leucine supply to the cells. These results support our previous findings that valyl-tRNA synthetase formation is subject to multivalent repression control by both isoleucine and valine. In contrast, repression control of iso-leucyl- and leucyl-tRNA synthetase formation is specifically mediated by the supply of the cognate amino acid.

Mechanism of an aminoacyl transfer ribonucleic acid synthetase

Mechanism of an aminoacyl transfer ribonucleic acid synthetase

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.

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.

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.

Effect of transfer ribonucleic acid on the rate law and mechanism of the adenosine triphosphate-pyrophosphate isotope exchange reaction of an aminoacyl transfer ribonucleic acid synthetase

The effect of tRNA on the kinetics of amino acid activation was investigated at pH 6, 17 °C, with the isoleucyl-tRNA synthetase from E. coli B. The terminal adenosine of the tRNA was removed in order to prevent its acceptance of amino acid. The amino acid activation reaction was monitored by the standard ATP-PP i isotope exchange assay. It was found that the modified tRNA partially inhibits the exchange reaction, as previously noted by others. By studying the reaction over a permutational variation in substrate concentrations, the rate law was obtained both in the presence and in the absence of saturating quantities of tRNA. In both cases, the same general rate law is obtained which suggests that the tRNA does not alter the mechanism of the amino acid activation reaction. An analysis of the parameters obtained from the rate laws shows that the binding of ATP to the naked enzyme and the equilibrium constant of the overall amino acid activation reaction are not altered by the tRNA. On the other hand, tRNA simultaneously weakens the binding of isoleucine and reduces the magnitude of a kinetic rate parameter. In addition to its role as a substrate, ATP was found to be a potent inhibitor of the reaction, regardless of the presence or absence of tRNA.

Binding of aminoacyl transfer ribonucleic acid synthetases to ribosomes from rabbit reticulocytes.

Binding of aminoacyl transfer ribonucleic acid synthetases to ribosomes from rabbit reticulocytes.

Divalent cations in transfer ribonucleic acid and aminoacyl transfer ribonucleic acid synthetase function and structure.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDivalent cations in transfer ribonucleic acid and aminoacyl transfer ribonucleic acid synthetase function and structureMichael Yarus and Stephen RashbaumCite this: Biochemistry 1972, 11, 11, 2043–2049Publication Date (Print):May 1, 1972Publication History Published online1 May 2002Published inissue 1 May 1972https://doi.org/10.1021/bi00761a008RIGHTS & PERMISSIONSArticle Views17Altmetric-Citations31LEARN 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 (758 KB) Get e-Alerts Get e-Alerts

Binding of complementary oligonucleotides to free and aminoacyl transfer ribonucleic acid synthetase bound transfer ribonucleic acid

Binding of complementary oligonucleotides to free and aminoacyl transfer ribonucleic acid synthetase bound transfer ribonucleic acid

Mutants of Escherichia coli unable to make protein at 42 C.

Members of a collection of mutants of Escherichia coli unable to form colonies on nutrient agar at 42 C have been characterized on the basis of their growth response to a shift from 32 to 42 C in liquid medium. Forty-four mutants, which show an abrupt, nonlethal cessation of growth when moved to the restrictive temperature, have been characterized with respect to the effect of the mutation responsible for temperature sensitivity on deoxyribonucleic acid, ribonucleic acid, and protein synthesis. In 12 mutants, the mutation causing temperature sensitivity of growth primarily affects protein synthesis, in each case through an altered aminoacyl-transfer ribonucleic acid synthetase. Mutants with temperature-sensitive glutamyl-, phenylalanyl-, and valyl-transfer ribonucleic acid synthetases have been obtained, and the genes specifying these enzymes have been mapped by conjugation and transduction. Another mutant has been shown to possess a temperature-sensitive tryptophanyl-transfer ribonucleic acid synthetase, but this is not responsible for inability to grow at 42 C on media containing tryptophan.

Stimulation of ribonucleic acid synthesis by chloramphenicol in a rel + aminoacyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.