tRNA Arg Research Articles

Transfer RNA selection at the ribosomal A and P sites

We have examined ribosomal selectivity among tRNAs using an equilibrium competition binding assay. Specifically, we evaluated the dissociation constants of tRNA Phe, tRNA Ile, tRNA fMet, tRNA Val, tRNA Arg, and tRNA Glu, for the A site and, separately, for the P site by measuring competition with [ 3H]phenyl-lactyl-tRNA Phe. We were able to distinguish A and P sites using the site-specific poisons, edeine and tetracycline. In the presence of poly(U), the affinity of tRNA Phe for the P site was significantly greater than that of any of the other five tRNAs. Moreover, the affinities of the tRNAs for the P site varied over three orders of magnitude and paralleled the predicted order of affinities based on potential codon-antidocon interactions. This strongly indicates coding capacity at the P site. In marked contrast, tRNA binding at the A site of poly(U)-programmed ribosomes indicated that tRNA Phe bound only ten- to 20-fold stronger than several non-complementary tRNAs, suggesting that the equilibrium properties of the bare ribosomal A site are very different from the P site. We propose that, in vivo, ribosomal contributions to the accuracy of protein synthesis include both specificity in tRNA selection at the A site and the coding capacity of the P site, as these sites are defined by in vitro criteria. Even at best, however, the precision of the ribosomes of the three strains we have examined is not better than the precision of unaided codon-anticodon interactions. This sets new constraints on the sources of accuracy in protein synthesis. We have also measured the relative affinities of tRNAs for the P site in the absence of message. By comparison of these with the codon-instructed P site, we conclude that message confers an additional affinity for a tRNA possessing a matching anticodon, rather than strongly rejecting tRNAs whose codons do not match. All the data we have are consistent with a large, almost non-specific affinity for tRNAs in both P and A sites, to which a smaller specific contribution is made by codon-anticodon base-pairing.

Neoplastic transformation-linked alterations in arginyl-tRNA synthetase activity

The behavior of the activity of arginyl-tRNA synthetase ( l-arginine: tRNA Arg Ligase(AMP-forming), EC 6.1.1.19) was determined in extracts of rat liver: normal adult, normal proliferating (from developing and from partially hepatectomized rats), and neoplastic (hepatomas of different growth rates) and in extracts of rat kidney cortex and transplantable kidney tumors. The K m values for arginine, ATP, and tRNA of the enzyme of the rapidly growing hepatoma 3924A were the same as those of the enzyme from the liver of control rats. The pH optima of the control and neoplastic livers were in the same range of 7.25–8.0. Taking the hepatic specific activity for arginyl-tRNA synthetase as 100%, deep layer of gut, thymus and testis had higher activity; renal cortex and spleen had the same activity; and skeletal muscle, brain, heart, lung, superficial layer of gut and adipose tissue had lower activity. In a wide spectrum of hepatomas of different growth rates, a significant increase of 1.4–2.4-fold in arginyl-tRNA synthetase activity was observed when compared with that of liver of control normal rats. This elevation in enzyme activity in hepatomas appears to be specific to neoplasia, since it is unaltered in regenerating and low in differentiating liver. The increase in arginyl-tRNA synthetase in the liver tumors appears to be transformation-linked, since the activity was increased in all hepatomas, even in the slowest growing ones. Furthermore, the increase in enzyme activity was not limited to hepatic neoplasms, since a rise was also observed in transplantable rat kidney tumors. Thus, the reprogramming of gene expression in neoplastic tissue entails an increase in arginine-tRNA synthetase activity.

M 7guanosine in tRNA of Escherichia coli

1. 1. The nucleoside content or six different individual tRNAs was re-investigated. 2. 2. Particular emphasis was placed on obtaining an accurate, quantitative estimation of the amount of the minor nucleoside m 7 guanosinc which was found to occur at less than one full residue in tRNA Phe( E. coli) and tRNA Arg( E. coli) but not in the other species of tRNAs examined. 3. 3. Several hypotheses to explain this observation are discussed.

The changes of tRNA Arg content and arginyl-tRNA synthetase activity in developing salmon testes during spermatogenesis

The changes of tRNA Arg content and arginyl-tRNA synthetase activity in developing salmon testes during spermatogenesis

Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA Arg3

At optimum magnesium concentration (10 mM) both yeast tRNA Arg 1 and tRNA Arg 3 are able to bind to poly(A,G) and A-G-A in presence of Escherichia coli ribosomes. With A-G-G only tRNA Arg 1 binds, whereas tRNA Arg 3 (anticodon mcm 5 U-C-U) is not bound. This result means that the methylcarboxymethyl substituant in position 5 of U prevents its wobble with G.

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

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA( N 2-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 tRNA Met f was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNA Met f is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the K m for S-adenosylmethionine is 3 μM and K i for S-adenosylhomocysteine is 0.11 μM for (guanine-1-)-methyltransferase. ( N 2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNA Phe, tRNA Val and tRNA Arg 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 tRNA Phe is maximally stimulated by 100 μM spermidine with a pH optimum of 8.0. ( N 2-Guanine-)-methyltransferase has a K m for S-adenosylmethionine of 2 μM and a K i for S-adenosylhomocysteine of 23 μM with E. coli K-12 tRNA as methyl acceptor.

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with 32P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.

Evidence that arginyl-adenylate is not an intermediate in the arginyl-tRNA synthetase reaction

Arginyl-tRNA synthetase has a reaction mechanism not typical of most aminoacyl-tRNA synthetases. It does not catalyze an amino acid-dependent ATP-PP 1 exchange in the absence of tRNA as do most enzymes of this class. In order to clarify the reaction mechanism by performing experiments with substrate levels of enzyme, we have modified the previous purification procedure. By the method presented, homogeneous enzyme can be prepared in approximately 10% yield. Pulse-labeling experiments indicate that no enzyme-bound arginyl-adenylate is formed in the absence of tRNA. Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNA arg. Two mechanisms compatible with these data are suggested.

Tertiary structure in E. coli tRNA Arg and tRNA Val

300 MHz proton NMR has been used to demonstrate that both Escherichia coli tRNA A rg and tRNA V al 1 have a tertiary structure base pair between 4-thiouridine (s 4U) at position 8 and A at position 14. Formation of this s 4U 8-A 14 base pair leads to a very low field (14.8 ppm) resonance in the spectra of both molecules. When s 4U is converted to U the 14.8 resonance is replaced by a new resonance which appears at 14.3 ppm. The presence of the tertiary structure s 4U 8-A 14 base pair greatly constrains the folding of these tRNAs in solution.

Primary structure of tRNA Arg II of E. coli B.

tRNA Arg II of E. coli has 77 nucleotides. There are eight minor nucleotides including inosine and 2-methyladenosine. Except for a few differences, the structure of tRNA Arg II is very similar to the structure of tRNA Arg I reported by Murao et al.3. The major difference is in the size of dihydrouridine loop. tRNA Arg II does not contain 2-thiocytosine. The unidentified nucleoside X seems to be a different modification other than nucleoside N reported to be present in tRNA Arg I.

Effect of sodium bisulfite modification on the arginine acceptance of E. coli tRNA Arg.

Escherichia coli tRNA Arg was treated with sodium bisulfite to convert exposed cytosine residues to uracil. This treatment resulted in the loss of amino acid acceptance of the tRNA Arg with pseudo first-order reaction kinetics. The active and inactive molecules were separated after about 60e active and inactive molecules were separated after about 60 percent inactivation and analyzed for U in various positions by finger-printing of the oligonucleotides produced by nucleases. The results show that C to U base transitions in the dihydrouridine loop and in the CCA terminus have no effect on the aminoacylation of this tRNA. Deamination of a cytosine residue at the second position of the anticodon resulted in the loss of amino acid acceptor activity of arginine transfer RNA.

Structure primaire des tRNA ArgIII de levure de bière : I. Hydrolyse totale par les ribonucléases pancréatique et T 1

Summary Brewer's yeast tRNA Arg III , 93–98 p. cent pure was prepared by countercurrent distribution followed by chromatography on a column of DEAE cellulose at 65° and by a reversed phase chromatography (RPC 1). It was completely digested with pancreatic ribonuclease and with T 1 ribonuclease. The separation and identification of the products were carried out by column chromatography followed by high voltage electrophoresis. The results of the analyses indicate that tRNA Arg III is in fact a mixture of at least two different subspecies, one of the differences being located in the 3′ terminal sequence. They both contain 12 minor nucleotides. A sequence of 21 nucleotides : A-C-U-xU-C-U-t 6 A-A-Ψ-C-A-G-A-A-G-A-hU-U-A-U-Gp has been deduced from the results obtained. The primary structure of the two tRNAs Arg III has been determined after partial digestion with pancreatic and T 1 ribonucleases as stated in the following paper.

Acylation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T − treated with chloramphenicol produces tRNA phe which is deficient in minor nucleosides. Undermodified tRNA phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA phe which was similar to the alteration of the purified tRNA phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA phe of phenoxyacetylated tRNA from late log phase E. coli 15T−. tRNA glu and tRNA Leu were not changed, but both tRNA Arg and tRNA Ile were altered. tRNA 2 Val and tRNA Met shifted in the elution profile; tRNA 1 Val and tRNA f Met were not affected. Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles. tRNA phe from E. coli 15T − treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA phe. tRNA phe from E. coli 15T −, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity. Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.

Raman studies of nucleic acids X. Conformational structures of Escherichia coli transfer RNAs in aqueous solution

Laser-excited Raman spectra of aqueous solutions of purified samples of Escherichia coli tRNA Glu, tRNA Arg, tRNA Val, tRNA fMet and tRNA 2 Phe are compared. The characteristic Raman frequencies and intensities, from scattering by symmetrical stretching vibrations of the phosphodiester groups, reveal that the total secondary structure of each tRNA is qualitatively and quantitatively the same. The percentage of nucleotide residues in each tRNA which exist in a highly ordered configuration is 84 ± 4 %, consistent with a cloverleaf structure provided two-thirds of the unpaired bases in loops are also in ordered configurations. The remaining nucleotides (16 ± 4 %) must occur in randomly oriented or highly-strained configurations of the tRNA chain. The Raman spectra also reveal specific differences in the amount of stacking of A and G residues in different tRNAs. These results provide strong evidence for the presence of specific tertiary structure (folding) in aqueous tRNA. A study of the melting transition of tRNA Glu over the range 32–90 °C reveals that appreciable order persists in the tRNA backbone at 90 °C and is attributable to residual stacking of the purines. Gross changes in the conformation of tRNA Val are also detected when excess Mg 2+ is present. The improved spectral sensitivity in this work demonstrates that the Raman spectrum of tRNA may be used to detect the presence of the minor nucleoside, dihydrouridine, and that the frequency region 1150–1450 cm −1 is highly sensitive to the base composition, base sequence and base-stacked secondary structure of tRNA.

The change in optical activity of amino acid-specific Escherichia coli transfer RNA containing 4-thiouridylate by chemical modifications

Circular dichroism (CD) spectra of five species of 4-thiouridine containing amino acid-specific tRNA from Escherichia coli were measured in the near ultraviolet region. In the 300–400 nm region, near 335 nm, which correlated with a minor absorption maximum, tRNA II Tyr showed a particular profile of CD having three bands, whereas all other tRNA (tRNA F Met, tRNA I Val, tRNA Phe, and tRNA Arg showed essentially similar spectra having two bands. This suggests that two adjacent 4-thiouridylate residues in tRNA II Tyr have quite different optical activity from the others due to the excitation splitting of the CD band by the nearest neighbor interaction. Thus, the CD profiles in the near ultraviolet region show dissymmetry near the thionucleotide in tRNA molecule. Oxidation of tRNA II Tyr with iodine showed a gross change of CD in the near ultraviolet region, the sign of which converted from positive to negative; tRNA F Met and tRNA I Val showed their characteristic bands in the near ultraviolet region after iodine oxidation. Treatment of tRNA with cyanogen bromide gave essentially similar profiles of CD as for the intact molecules. Treatment with S-benzylthioisothiourea resulted in an unique CD spectrum for tRNA II Tyr, just a reversed profile of its iodine oxidation. No significant change in CD in the ultraviolet region was detected even after subjecting these tRNA to the three previously mentioned chemical modifications.

Indospicine as an arginine antagonist in Escherichia coli and Pseudomonas aeruginosa

Under conditions leading to low intracellular concentrations of arginine indospicine (L-2-amino-6-amidinohexanoic acid) inhibits the growth of Escherichia coli and Pseudomonas aeruginosa. Indospicine is charged on tRNA Arg and incorporated into bacterial protein. In vitro it inhibits the activity of N- acetylglutamate 5-phosphotransferase, the feedback sensitive enzyme of arginine biosynthesis in Pseudomonas aeruginosa.

Isolation and characterization of 2-methyladenosine from Escherichia coli tRNA Glu2, tRNA Asp1, tRNA His1 and tRNA Arg

A minor component was isolated from Escherichia coli tRNA Glu 2, tRNA Asp 1, tRNA His 1 and tRNA Arg and identified as 2-methyladenosine. 2-Methyladenylic acid was obtained by mild treatment of tRNA with ribonuclease and subsequently converted to the corresponding nucleoside and to the base by treatment with E. coli alkaline phosphomonoesterase and by acid hydrolysis, respectively. The nucleotide, nucleoside and base thus obtained were identified with the corresponding authentic samples by comparison of their ultraviolet absorption spectra and mobilities on thinlayer chromatography in several solvent systems. This is the first demonstration of the presence of 2-methyladenosine in purified tRNA's.

Studies on the mechanism of repression of arginine biosynthesis in Escherichia coli: IV. Further studies on the role of arginine transfer RNA repression of the enzymes of arginine biosynthesis

Two column Chromatographic procedures have been utilized to purify and fractionate arginyl-transfer RNA: DEAE-Sephadex and reverse-phase column chromatography. Five iso-accepting species of tRNA Arg were found and they recognize the six arginine codons. Similar elution profiles of arginyl-tRNA species were obtained when tRNA was extracted from the wild-type Escherichia coli K12 strain grown under conditions of repression and derepression. In a more sensitive system using an arginyl-tRNA synthetase mutant with a low level of in vivo tRNA Arg charging, no effect of repression on the profiles of the charged arginyl-tRNA was found. These results indicate that significant variations in tRNA Arg as a result of repression of the arginine biosynthetic enzymes are unlikely.

Binding of transfer RNA and arginine to the arginine transfer RNA synthetase of Escherichia coli

The arginine transfer RNA synthetase of Escherichia coli is protected against thermal denaturation by its substrates arginine and tRNA Arg but not by ATP, alone or in combination with the other substrates. The protection given by transfer RNA alone requires 20-fold higher concentrations above pH 7 than below, whereas the protection by arginine requires less at higher pH values but does not show a sharp transition at any pH value. Modification of the 3′-terminal adenosine eliminates catalytic activity but does not affect the binding or degree of protection. Stepwise removal of groups from the 3′-hydroxyl end of the transfer RNA shows that the three terminal nucleotides do not participate significantly in binding but that the phosphate that links the cytidine residues plays a role in increasing the resistance of the protein to denaturation. The concentration of arginine required to protect the arginine transfer RNA synthetase is decreased by the presence of tRNA Arg. In contrast to the protection by the tRNA alone, the “synergistic protection” requires that the 3′-adenosine be intact. Esterification of arginine to transfer RNA does not increase the protection given by the RNA alone and eliminates synergistic protection.