Glutamyl-tRNA synthetases from wheat. Isolation and characterization of three dimeric enzymes

Three dimeric glutamyl-tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts. The chloroplast enzyme (Mr = 110 000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity. Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GluRS E, has eukaryotic (wheat germ) tRNA specificity. Both enzymes exhibit a molecular weight close to 160 000. Each of these enzymes co-eluate on hydroxyapatite and phosphocellulose chromatographies with an unstable active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers.     

Isolation and characterization of a tRNA(guanine-7-)-methyltransferase from Salmonella typhimurium

The tRNA modifying enzyme, S-adenosylmethionine:tRNA(guanine-7-)-methyltransferase, has been extensively purified from Salmonella typhimurium. A rapid and efficient purification method using phosphocellulose chromatography followed by ammonium sulfate precipitation and Sephadex G-100 gel filtration is described. The enzyme appears to be a single polypeptide chain with a molecular weight of approximately 25 000--30 000 daltons. The Km for S-adenosylmethionine and for undermethylated tRNA is 53 microM and 3.4 microM, respectively. The methylation reaction is dependent on added monovalent or divalent cations; 5 mM spermidine, 3 mM MgCl2 and 1 mM spermine are the most effective. The enzyme, though not homogeneous, is free from contaminating ribonucleases and other tRNA methyltransferases.     

Proteolytic cleavage of methionyl transfer ribonucleic acid synthetase from Bacillus stearothermophilus: effects on activity and structure

Methionyl-tRNA synthetase from Bacillus stearothermophilus, a dimer of molecular weight 2 X 85K, is converted by limited subtilisin digestion into a fully active monomeric fragment of molecular weight 64K. The reversible methionine activation reaction of these enzymes was followed through the variation of the intensity of their trypotophan fluorescence. Equilibrium and stopped-flow experiments show that the rate and mechanism for adenylate formation supported by the monomeric derivative are undistinguishable from those of each adenylating site of the native dimeric enzyme. In contrast, the rate of tRNA aminoacylation is improved upon limited proteolysis of the native enzyme. This behavior can be related to the anticooperativity of the binding of tRNA molecules to native dimeric enzyme. Accordingly, at 25 degrees C, the dimer might behave as a half-of-the-sites enzyme with only one active tRNA site at a time, compared to two after limited proteolysis with consequent irreversible disociation into two 64K fragments. Another modified form of the enzyme is obtained through limited tryptic digestion. This derivative is completely devoid of activity although its molecular weight under nondenaturating conditions remains undistinguishable from that of the 64K fragment generated by subtilisin. Denaturation reveals that this tryptic derivative is composed of two subfragments with molecular weights of 33K and 29K, respectively. The same fragments may also be directly obtained through limited tryptic digestion of the subtilsic fragment. Interestingly, although trypsin treatment has abolished the activity of the enzyme, fluorescence studies demonstrate that the ATP and methionine binding sites have remained intact. It is shown that the effect of the internal cut made by trypsin into the active 64K fragment has been to considerably depress the "coupling" between the methionine and nucleotide binding sites. Finally, the rate of inactivation of the enzyme by trypsin is observed to be substantially decreased by in situ synthetized methionyl adenylate but not by tRNA. These properties and others are discussed in relation to the problem of its significance of repeating sequences and structural "domains" within the class of aminoacyl-tRNA synthetases.     

Subunit structure and tRNA-binding properties of Bombyx mori Glycyl-tRNA synthetase

Large amounts of glycyl-tRNA synthetase were purified from the posterior silk glands of Bombyx mori. The synthetase was estimated to be a dimer with a molecular weight of 180,000. When the enzyme solution was diluted, the dimer dissociated into monomers which were inactive in tRNA aminoacylation. The aminoacylation was investigated with two isoaccepting tRNAsGly isolated from the posterior silk glands. Transfer RNA1Gly was aminoacylated 2-fold faster than tRNA2Gly. Transfer RNA-binding experiments revealed that tRNA1Gly binds with the enzyme in a molar ratio of 2:1, whereas tRNA2Gly formed a 1:1 complex with the enzyme. Based on these experimental results, we proposed that the Bombyx mori glycyl-tRNA synthetase has two active sites for tRNA aminoacylation and that the number of tRNA molecules bound on the synthetase closely correlates with the velocity of aminoacylation.     

Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli

The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.     

tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8

tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8. The specific activity of the enzyme is about 50 000 and the yield of activity more than 20%. The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme. The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000. By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000. These data allow to suggest a subunit structure of the enzyme. The enzyme is highly thermostable and is most active at 80 degrees C. The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.     

Purification and characterization of a tRNA methylase from Salmonella typhimurium.

A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.     

N 6 -( 2 -isopentenyl) adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli

An enzyme has been partially purified from Escherichia coli which catalyzes in vitro the transfer of the Δ²-isopentenyl group from Δ²-isopentenyl pyrophosphate to an adenosine residue in Mycoplasma sp. (Kid) tRNA. The product of the reaction is N⁶-(Δ²-isopentenyl) adenosine, which is known to be absent in this Mycoplasma tRNA. The enzyme has an approximate molecular weight of 55,000 daltons, requires reduced sulfhydryl groups and a divalent metal ion for full activity, and is specific for tRNA.     

Ultracentrifugation studies of yeast valyl-tRNA synthetase and of its interaction with tRNAVal.

Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 +/- 1500 and a sedimentation coefficient (SO 20, w) of 6.3 +/- 0.3 were found for the native enzyme. When the enzyme (3--60 muM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amounts of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA/enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant from tRNA/enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0--7.5) and in various buffers.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Purification and some properties of the histidyl-tRNA synthetase from the cytosol of rabbit reticulocytes.

The histidyl-tRNA synthetase of rabbit reticulocyte cytosol has been purified 84 000-fold to apparent homogeneity with a specific activity of 687 nmol of histidyl-tRNA formed per min per mg of protein. Ten to 15% of the enzyme activity is sedimented with the ribosomes while the remainder is in the cytosol. The purified enzyme has a molecular weight of 122 000 as determined by sucrose density gradient centrifugation. Gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate suggests that it is composed of two similar subunits with a molecular weight of approximately 64 000. The enzyme has a magnesium optimum of 45 mM; however, this is reduced to 5 mM in the presence of an intracellular potassium concentration (160 nM). The enzyme acylates the two histidine tRNA isoacceptors of rabbit reticulocytes with similar Km values and at similar rates.     

A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNA(Val) from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNA(Val) to valyl-tRNA synthetase from B. stearothermophilus were determined between 5 degrees and 60 degrees C.     

Purification and characterization of Escherichia coli RNase T

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.     

Purification and properties of leucyl-tRNA synthetase from the cow mammary gland

Three forms (E1, E2 and E3) of leucyl-tRNA synthetase (LeuRS) were separated by DEAE-cellulose chromatography of total aminoacyl-tRNA synthetases from cow lactating mammary gland. The method of purification of all three components is described. E1 is a dimeric molecule (alpha 2) of molecular weight 182 000. Two other forms of molecular weight 67 000 and 64,000 consist of a single polypeptide chain as determined by polyacrylamide gel electrophoresis. Optimum conditions and kinetic parameters of leucyl-tRNA formation were studied for every enzyme form. The low values of Vmax and thermostability are characteristic of E3. All forms of LeuRS interact with 6 isoaccepting tRNA(Leu) from lactating mammary gland and can activate leucine in the absence of tRNA. E2 and E3 are supposed to derive from the native enzyme by endogenous proteolysis. The physico-chemical properties of native LeuRS from lactating mammary gland are compared with those of LeuRS's from other sources.     

Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA(N2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1-)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNAMetf was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNAMetf is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the Km for S-adenosylmethionine is 3 micrometer and Ki for S-adenosylhomocysteine is 0.11 micrometer for (guanine-1-)-methyltransferase. (N2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNAPhe, tRNAVal and tRNAArg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5'-terminus of these tRNAs. The methylation of tRNAPhe is maximally stimulated by 100 micrometer spermidine with a pH optimum of 8.0. (N2-Guanine-)-methyltransferase has a Km for S-adenosylmethionine of 2 micrometer and a Ki for S-adenosylhomocysteine of 23 micrometer with E. coli K-12 tRNA as methyl acceptor.     

Modification of phenylalanyl-tRNA synthetase from baker's yeast by proteolytic cleavage and properties of the trypsin-modified enzyme.

Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, alpha of molecular weight 73000 and beta of molecular weight 63000. The enzyme is an asymmetric tetramer alpha-2beta-2, which binds two moles of each ligand per mole. Incubation of the purified enzyme with trypsin results in an irreversible conversion: the alpha-subunit remains apparently unchanged but beta is rapidly degraded and yields a lighter species beta of molecular weight 41000. The trypsin-modified enzyme is an alpha-2beta-2 molecule which can still activate phenylalanine but cannot transfer it to tRNA-Phe; furthermore it does not bind tRNA-Phe but its kinetic parameters are identical to those of the native enzyme with respect to ATP and phenylalanine. Therefore the two beta subunits play a critical part in tRNA binding. Isolated alpha or beta subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation.     

Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.     

Purification and properties of tRNA(adenine-1)-methyltransferase from rat liver.

An S-adenosylmethionine-dependent tRNA(adenine-1)-methyltransferase has been purified 8,000-fold from rat liver. This preparation gives a single band on polyacrylamide gel electrophoresis and is stable in long term storage. The enzyme has a molecular weight of approximately 95,000. The single methylating capacity of this adenine-1 methyltransferase, using Escherichia coli tRNA2Glu, is methylation of the invariant adenine in the GTpsiC loop. The methylation reaction is dependent on added cation with 20 to 40 mM putrescine being most effective. The Km for S-adenosylmethionine was found to be 0.3 micron, while the Ki for the product inhibitor S-adenosylhomocysteine was 0.85 micron. The Km for tRNAMetf is 12 nM while that for tRNAGlu2 is 33 nM.     

Reactions at the 3' terminus of transfer ribonucleic acid. IX. tRNA nucleotidyltransferase activity in bacteriophage T4-infected Escherichia coli

There was no detectable increase in tRNA nucleotidyltransferase activity upon infection of $\text{Escherichia coli}$ A19 with bacteriophage T4. Three mutant strains which contained low levels of tRNA nucleotidyltransferase activity also showed no increase in activity after infection. tRNA nucleotidyltransferase was purified from both uninfected and T4-infected cells and examined for possible modification. It was found that enzyme purified from both types of cells eluted from DEAE cellulose at the same specific conductivity. In addition, the molecular weight of tRNA nucleotidyltransferase purified from both uninfected and T4-infected cells was approximately 45,000 daltons as determined by chromatography on Sephadex G-100. These results suggest that T4-infection does not lead to synthesis of a new virus-specific tRNA nucleotidyltransferase nor does it cause modification of the host enzyme.