Valyl-transfer Ribonucleic Acid Synthetase Research Articles

.beta.- and .gamma.-Thio analogs of adenosine triphosphate as probes of the Escherichia coli valyl transfer ribonucleic acid synthetase reaction pathway. A novel stereospecific interchange of adenosine 5'-O-(2-thiotriphosphate) to adenosine 5'-O-(3-thiotriphosphate)

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT.beta.- and .gamma.-Thio analogs of adenosine triphosphate as probes of the Escherichia coli valyl transfer ribonucleic acid synthetase reaction pathway. A novel stereospecific interchange of adenosine 5'-O-(2-thiotriphosphate) to adenosine 5'-O-(3-thiotriphosphate)Edward F. Rossomando, Linda Tombras Smith, and Mildred CohnCite this: Biochemistry 1979, 18, 25, 5670–5674Publication Date (Print):December 11, 1979Publication History Published online1 May 2002Published inissue 11 December 1979https://doi.org/10.1021/bi00592a023RIGHTS & PERMISSIONSArticle Views40Altmetric-Citations15LEARN 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 (659 KB) Get e-Alerts Get e-Alerts

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of valyl-transfer ribonucleic acid synthetase

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile valyl-transfer ribonucleic acid (tRNA) synthetase were selected for growth at 40 degrees C. Of these, a large number still contain the thermolabile valyl-tRNA synthetase. Three of these revertants contained an increased level of the thermolabile enzyme. The genetic locus, valX, responsible for the enzyme overproduction, is adjacent to the structural gene, valS, of valyl-tRNA synthetase. Determination (by radioimmunoassay) of the turnover rates of valyl-tRNA synthetase showed that the increased level of valyl-tRNA synthetase is due to new enzyme synthesis rather than decreased rates of protein degradation.

In vivo regulation of chromosomal beta-lactamase in Escherichia coli

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.

Purification and properties of a T4 bacteriophage factor that modifies valyl-tRNA synthetase of Escherichia coli.

After T4 bacteriophage infects Escherichia coli, a peptide tau, produced under the control of a phage gene, binds to the host valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) and thereby changes several of its physicochemical properties. The interaction of tau with the host enzyme was investigated in vitro after extensively purifying the factor from T4-infected E. coli using a rapid purification procedure. The tau preparation migrated as a single, protein-staining band with a molecular weight of 11,000 during sodium dodecyl sulfate-gel electrophoresis. The purified peptide completely converted partially purified valyl-tRNA synthetase from uninfected E. coli into the form present in cell-free extracts prepared from virus-infected bacteria. The enzyme modified in vitro also exhibited the enhanced affinity for tRNA characteristic of the viral form of valyl-tRNA synthetase. The addition of bulk tRNA from E. coli B, tRNAVal, or tRNA1Val to enzyme modified in vitro increased its sedimentation rate to that of enzyme prepared from phage-infected cells. Amino acid analysis of the purified tau peptide revealed a relatively high concentration of the amino acids lysine and alanine, and a lack of detectable proline, tyrosine, phenylalanine, and methionine.

Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli

The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.

The role of divalent cations in the reactions of valyl transfer ribonucleic acid synthetase of Escherichia coli. Effects of spermine and ethylenediaminetetraacetate.

We have analyzed the function of spermine in the aminoacylation of tRNA-Val by the valyl-tRNA synthetase of Escherichia coli. Our results indicate that Mg2+ is required for the aminoacylation reaction as well as for the ATP-PP-i exchange catalyzed by this enzyme. The apparent stimulation by spermine is a function of the tRNA used, which appears to contain bound cations even after dialysis against 10 minus 4 M EDTA. Higher concentrations of EDTA totally abolish spermine-stimulated esterification of tRNA-Val.

The infrastructure of valyl-transfer Ribonucleic Acid synthetase from yeast.

Valyl-tRNA synthetase from yeast is shown to contain a significant proportion of repeated sequence, similar to the enzyme from bacterial sources.

The Effect of Transfer Ribonucleic Acid on Virally Modified Valyl Transfer Ribonucleic Acid Synthetase of Escherichia coli

Abstract The valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) of Escherichia coli and the modified form of the enzyme that appears after T4 bacteriophage infection were analyzed by sucrose density gradient electrophoresis. Work with cell-free extracts and purified enzymes indicates that tRNA from E. coli B specifically accelerates the electrophoretic mobility of bacteriophage-modified valyl-tRNA synthetase. While the purified, modified form of the enzyme is intrinsically more stable to urea denaturation than the normal enzyme, tRNA accentuates the difference. Finally, tRNA specifically increases the sedimentation rate of purified, modified valyl-tRNA synthetase in sucrose density gradients. As a phage-directed component τ can be shown to be specifically associated with the modified form of the enzyme; we think the component is responsible for the unique effects of tRNA on bacteriophage-modified valyl-tRNA synthetase.

Metabolic Regulation of the Arginyl and Valyl Transfer Ribonucleic Acid Synthetases in Bacteria

Abstract The arginyl and valyl transfer ribonucleic acid synthetases (EC 6.1.13 and EC 6.1.1.9, respectively) in various strains of Escherichia coli and Salmonella typhimurium were measured over a wide range of steady state growth rates. Both enzymes were found to increase approximately 2.5-fold in specific activity over a 7-fold increase in growth rate. Appropriate tests indicated that this change was not the result of stimulation or inhibition of enzyme activity. Use of antibodies prepared against purified valyl-tRNA synthetase revealed differences in the amount of this enzyme in cell-free extracts of cultures grown at different growth rates. By use of a sequential labeling technique it was possible to demonstrate that the low activity of these two enzymes in extracts from slow growing cultures was not caused by specific degradation. During transitions in growth rates brought about by shifts in media, the activity of each of these enzymes changed similarly to the accumulation of RNA, and not in a manner one would predict for enzymes regulated by amino acid supply. By sequential labeling it was shown that the increased differential rate of activity accumulation seen for these enzymes after a shift up was the result of an increased differential rate of synthesis. The pattern of regulation of the arginyl- and valyl-tRNA synthetases seen in steady state cultures and during growth rate transitions has been termed metabolic regulation. The regulation of these two enzymes is similar under these conditions to that of other members of the bacterial cell's protein synthesizing machinery, and is difficult to reconcile with an amino acid mediated repression of these enzymes.

Accumulation of guanosine tetraphosphate in T7 bacteriophage-infected Escherichia coli.

Accumulation of guanosine tetraphosphate (ppGpp) in Escherichia coli strain AS19 valS, carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and infected with bacteriophage T7, was studied. Valine starvation was achieved by culturing this strain at 42 C. Addition of rifampin to an uninfected culture at the nonpermissive temperature resulted in loss of accumulated ppGpp; however, cultures infected with phage T7, treated with rifampin, and then shifted to the nonpermissive temperature maintained the ability to accumulate ppGpp. Moreover, treatment of the T7-infected culture with rubidomycin, an antibiotic which inhibits transcription, did not reduce the amount of ppGpp accumulated following shift to the nonpermissive temperature. Measurements of the instantaneous rate of T7 transcription showed that it is not under stringent control of amino acids. ppGpp synthesized in T7-infected E. coli appears to be more stable than its counterpart in an uninfected culture. These results are interpreted to mean that ppGpp production is not directly dependent on transcription and arises instead from inhibition of another reaction, most likely some aspect of translation.

Growth-linked instability of a mutant valyl-transfer ribonucleic acid synthetase in Escherichia coli.

The valyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli strain NP2907, previously described as having an elevated K(m) for adenosine triphosphate and reduced stability in vitro compared to the wild type, was found to be conditionally thermolabile in vivo. The rate of inactivation of this enzyme at a particular temperature appears to be coordinated with the rate of growth; at 40 C this coordination results in equal rates of synthesis and destruction over a wide range of growth rates. In vitro studies showed that conditions favoring maintenance of the valyl-tRNA synthetase-valyl adenylate complex conferred complete protection against inactivation at 40 C, whereas the further addition of uncharged tRNA caused rapid, irreversible decay. We propose that the rate of inactivation of this mutant valyl-tRNA synthetase in vivo is a function of the ratio of deacylated to acylated tRNA(val) and that this ratio is a function of growth rate. The event which renders the valyl-tRNA synthetase susceptible to inactivation is likely to be the normal breakdown of the valyl-tRNA synthetase-valyl-adenylate complex during a cycle of aminoacylation of tRNA(val).

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.

A Comparative Study of the Interactions of Escherichia coli Leucyl-, Seryl-, and Valyl-Transfer Ribonucleic Acid Synthetases with Their Cognate Transfer Ribonucleic Acids

Abstract Kinetic and equilibrium binding studies (enzyme-tRNA complex formation examined by gel filtration) were conducted in the same buffer with leucyl-tRNA synthetase and five tRNAleu isoacceptors, seryl-tRNA synthetase and three tRNAser isoacceptors, and valyl-tRNA synthetase and two tRNAval isoacceptors. All of the enzymes and tRNAs were highly purified from one strain of Escherichia coli K-12. The tRNA Km values and relative binding affinities are closely similar (estimates of ΔF range between 10 and 11 kcal per mole), suggesting that tRNA recognition by these three enzymes has some common thermodynamic feature. The results at least indicate basic similarities in the geometry of the aminoacyl-tRNA synthetase-tRNA complex. This notion is supported by the finding that all three enzymes exhibit a reaction sequence of the bi uni uni bi ping pong type, the addition order of substrates being amino acid, ATP, tRNA. The three enzymes differ in their susceptibility to ATP substrate inhibition.

Structure and Function of Transfer Ribonucleic Acid: IV. COMPLEXES BETWEEN VALYL TRANSFER RIBONUCLEIC ACID SYNTHETASE AND STRUCTURALLY MODIFIED TRANSFER RIBONUCLEIC ACID SPECIFIC FOR VALINE

Abstract The formation of a stable complex between valyl transfer ribonucleic acid synthetase from yeast and transfer RNA specific for valine (tRNAval) from the same source has been used as a model for the recognition between enzyme and tRNA. With the use of sucrose gradient centrifugation to isolate the complex, the major fraction of tRNAval (tRNA1val) was found to retain its ability to form a stable complex with the enzyme even after a digestion with snake venom phosphodiesterase that had removed as much as an average of 7 nucleotide residues from the 3'-hydroxyl end. The digested tRNAs also showed competitive inhibition of complex formation with native tRNA1val and the relative affinities of the enzyme for the different tRNA1val preparations that could be calculated from the inhibition experiments showed that this extensive modification at the 3'-hydroxyl end had not lowered the affinity of the enzyme for the modified tRNA1val compared to native tRNA1val by more than a factor of 2.

Structure and Function of Transfer Ribonucleic Acid: III. Some Properties of a Complex Between Valyl Transfer Ribonucleic Acid Synthetase and Transfer Ribonucleic Acid Specific for Valine

Abstract The formation and some properties of a stable complex between valyl transfer ribonucleic acid synthetase and transfer RNA from yeast have been investigated by three different methods for the isolation of the complex: filtration over Sephadex, sucrose gradient centrifugation, and electrophoresis on Pevikon as supporting medium. Transfer RNA specific for valine (tRNAval) can be separated into two fractions, tRNAvalI and tRNAvalII, in the approximate proportions 3:1 by chromatography on DEAE-cellulose. With the Sephadex filtration technique only the minor fraction, tRNAvalII, gave a stable complex with the enzyme. When sucrose gradient centrifugation or electrophoresis was used it was possible to show the formation of stable complexes between the enzyme and both tRNAvalI and tRNAvalII. With the centrifugation technique complexes between the enzyme, AMP, valine, and valyl-tRNA could also be shown. This technique gave a separation of complex and free enzyme that permitted a determination of the molar ratio of tRNAval to enzyme in the complex. The values obtained ranged from 0.7 to 0.9, indicating one tRNA-site per enzyme molecule. The centrifugation data also showed that only 50 to 60% of the enzyme molecules had the ability to form a stable complex with tRNAvalI while all of the enzyme could be obtained as a complex with tRNAvalII. tRNAval when present as a complex with the enzyme was protected against T1-RNase under conditions in which free tRNAval was completely inactivated. The protection against phosphodiesterase, although significant, was much less pronounced.

Mapping of a structural gene for valyl-transfer ribonucleic acid synthetase in Escherichia coli by transduction.

A structural gene, valS, for the valyl-transfer ribonucleic acid synthetase of Escherichia coli has been mapped on the clockwise side of pyrB and is closely linked to it.