Phosphorylation. IV. Selective phosphorylation of the primary hydroxyl group in nucleosides

Imai, K.; Fujii, Shoichiro; Takanohashi, Kunio; Furukawa, Yoshiyasu; Masuda, Tohru; Honjo, Mikio

doi:10.1021/jo01258a004

Cited by 74 publications

(38 citation statements)

References 4 publications

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“…3'-Deoxyadenosine was phosphorylated (7) and converted to the 5'-triphosphate (8) by known methods. Purified venom phosphodiesterase was obtained from Boehringer Mannheim; DEAE cellulose and GF/A glass fiber discs were from Whatman.…”

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confidence: 99%

Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Hecht¹,

Chinualt²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Transfer RNAs terminating in 2'-or 3'-deoxyadenosine were prepared from unfractionated E. coil and yeast (Saccharomyces cerevisiae) tRNAs and purified to remove unmodified tRNAs. The modified tRNA species were assayed for aminoacylation with each of the 20 amino acids to determine the initial position of tRNA aminoacylation. The E. coli and yeast aminoacyl-tRNA synthetases specific for arginine, isoleucine, leucine, methionine, phenylalanine, and valine, as well as the E. coil glutamyl-tRNA synthetase, aminoacylated only those cognate tRNAs terminating in 3'-deoxyadenosine (i.e., those having a 2'-OH group). On the other hand, those E. coil and yeast synthetases specific for alanine, glycine, histidine, lysine, proline, serine, and threonine, as well as the yeast synthetase specific for glutamine, utilized exclusively those tRNAs having an available 3'-OH group on the 3'-terminal nucleoside, while the E. coil and yeast synthetases specific for asparagine, cysteine, and tyrosine, and the yeast aspartyl-tRNA synthetase, utilized both of the modified cognate tRNAs. The only observed difference in specificity between the E. coli and yeast systems was for tRNATrP, which was aminoacylated on the 2'-position in E. coli and the 3'-position in yeast. The results indicate that the initial position of aminoacylation is not uniform for all tRNAs, although for individual tRNAs the specificity has been conserved during the evolution from a prokaryotic to eukaryotic organism. Although aminoacyl-tRNA undoubtedly exists in solution as a rapidly equilibrating mixture of the 2'-and 3'-O-aminoacyl species, a description of the initial position of aminoacylation of individual tRNAs by their cognate aminoacyl-tRNA synthetases is of interest as part of a complete description of the precise mechanism of peptide bond formation. Several reports have described the preparation of modified tRNAs terminating in 2'-and 3'-deoxyadenosine and 2'-and 3'-Omethyladenosine and the utilization of those tRNAs in certain of the partial reactions of protein biosynthesis (1-4). Substrate activity of the modified tRNAs in phenylalanine acceptor assays was limited to those species terminating in 3'-deoxyadenosine and 3'-O-methyladenosine; the latter was shown to be aminoacylated poorly relative to unmodified tRNA, also indicating an apparent steric requirement in the aminoacylation reaction. More recently a report has appeared describing the substrate activity of a number of Escherichia coli tRNAs terminating in 2'-and 3'-deoxyadenosine (5) and some of the conclusions were supported by a related study utilizing tRNAs terminating in 2'(3')-amino-2'(3')-deoxyadenosine (6).Abbreviations: tRNA-C-COH, tRNA missing the 3'-terminal adenosine moiety; DBAE-cellulose; N-[N'-(m-dihydroxyborylphenyl)succinamyllaminoethyl-cellulose; Ax unit, the amount of material giving an A of 1.0 at wavelength X when dissolved in 1 ml of solution and measured in a cell with a 1 cm light path; nucleoside Q, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine...

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mentioning

confidence: 99%

Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Hecht¹,

Chinualt²

1976

Proc. Natl. Acad. Sci. U.S.A.

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“…Phosphoenolpyruvate and pyruvate kinase were obtained from Sigma Chemicals, as were CTP, ATP, GTP, GDP, 2'-dATP, and cordycepin [which was converted to 3'-dATP chemically (19,20)]. Aminoethyl-cellulose (Sigma) was converted to (acetylated) DBAE-cellulose recovered by elution with 50 mM Na+-Mes [sodium 2-(Nmorpholino)ethanesulfonate], pH 5.5, containing 1 M NaCI.…”

Section: Methodsmentioning

confidence: 99%

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

Hecht

Tan

Chinault

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2'-0-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay [3Hlaminoacyl-tRNAs from Escherichia coli terminating in 2'-or 3'-deoxyadenosine for binding to EF-Tu to determine the possible positional specificity of the factor. Binding of modified aminaoc l-tRNAs to EFTuGTP was measured both as a function of the ability of EFTuGTP to diminish the rate of chemical deacylation of[3H]aminoacyl-tRNAs and by gel filtration of the individual ternary complexes. Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including three (tRNAAsP, tRNACYs, and tRNATYr) for which isomeric modified aminoacyl-tRNAs were available. All of the modified aminoacyltRNAs were protected from deacylation, although generally to a lesser extent than the corresponding unmodified species.Six modified tRNA isoacceptors (including tRNATrP and tRNATYr, fQr which both modified aminoacyl-tRNAs were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu-GTP complexes. These experiments were also done in the presence of equimolar amounts of the corresponding unmodified [14Claminoacyl-tRNAs, and the relative affinities for a limiting amount of EF-TuGTP were measured. The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positionalisomers of aminoacyl-tRNA with no

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“…The assay mixture (80 pul) contained 50 mM Tris hydrochloride (pH 8.0), 5 mM MgCl2, 150 mM KCl, 5 mM dithiothreitol, and labeled deoxynucleoside triphosphate. The template-primer concentration for [PH] thymidine incorporation was 15 ,uM poly(A)-oligo(dT) [12][13][14][15][16][17][18] (concentration in nucleotide base pairs); for [3H]deoxyguanosine incorporation, the concentration of poly(C)-oligo (dG)12_18 was 188 puM in nucleotide base pairs. The concentrations of the template primers were chosen to be saturating for the enzymes.…”

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Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses

Chernow

Boehme

et al. 1988

Antimicrob Agents Chemother

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Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.

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Phosphorylation. IV. Selective phosphorylation of the primary hydroxyl group in nucleosides

Cited by 74 publications

References 4 publications

Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Position of aminoacylation of individual Escherichia coli and yeast tRNAs.

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses

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