Chemical Modifications of Transfer RNA Species. Desulfurization with Raney Nickel

Abstract

A single, modified nucleoside in Escherichia coli tRNA, 6-(3-methyl-2-butenylamino)-2-methylthio-9-#-D-ribofuranosylpurine, has been desulfurized with Raney nickel to afford its probable biosynthetic precursor. The limitations of the reaction at the nucleoside and tRNA levels and its lack of inhibition of the amino acid acceptor activity of tRNA are described.Certain modified nucleosides found in tRNA are also potent plant growth hormones (1). Where these hormones, or cytokinins, occur in tRNA, they occupy only that position adjacent to the 3'-end of the anticodon triplet, and only in tRNA species that respond to codons beginning with uridine (1, 2). Four cytokinins (1-4) have been isolated as components of tRNA (1-3).To facilitate the study of the metabolic function and biosynthesis of one of these modified nucleosides, 6-(3-methyl-2-butenylamino)-2-methylthio-9-,3-D-ribofuranosylpurine (3), a procedure has been developed for hydrogenolysis of the methylthio groups of 3, where it occurs in Escherichia coli tRNA.The identification of cytokinins as components of tRNA has prompted considerable research into the metabolic importance of these compounds. Studies on E. coli tRNA, for example, indicated that cytokinins were located adjacent to the 3'-end of the anticodons in tyrosine (4) and phenylalanine (5) tRNAs, and were also present, presumably adjacent to the anticodon, in cysteine, tryptophan, serine, and leucine tRNAs (6, 7). Gefter and Russell (8) also presented evidence that the cytokinins were important in the specific binding of tRNA to the appropriate ribosome-bound codon triplets, and Fuller and Hodgson (9) hypothesized the importance of this group to proper codon-anticodon interaction in protein synthesis.Peterkovsky (10) and Hall (11) showed that the mevalonate-requiring bacteria Lactobacillus acidophilus and Lactobacillus plantarum rapidly incorporated [14C Imevalonate into the side chain of 6-(3-methyl-2-butenylamino)-9-j3-Dribofuranosylpurine (4) in tRNA. Hall (12-14) later showed that tRNA, which had been partially modified by treatment with permanganate, incorporated [14C]A2-isopentyl pyrophosphate to a greater extent than unmodified tRNA. In addition, Miura and Miller (15) demonstrated that the fungus Rhizopogon roseolus contained the enzymes necessary to convert exogenous 6-(3-methyl-2-butenylamino)purine (4, purine portion) to the hydroxylated derivative 2. Prior to the identification of 3 as a component of E. coli tRNA, Goodman et al. (4) showed that the nucleoside adjacent to the anticodon of E. coli tRNATVT, now assumed to be 3, could be labeled with both [n5S]sulfate and [methyl-14C1-methionine. Rosenberg and Gefter (16) presented evidence consistent with an iron dependence for the in vivo formation of 3 from 4 in tRNA. After the identification of 3 as a tRNA component (17)(18)(19), Harada et al. (19) suggested that this nucleoside must be formed by the modification of 4 in one or two steps.To test for the presence of an enzyme(s) that would modify 4 to 3 at the tRNA level, and to...

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

“…1. Molecular masses and 1 H-NMR data of the synthesized compounds agreed with reported values (Bressi et al, 2000;Hecht et al, 1971;Véliz & Beal, 2000;Vittori et al, 2000).…”

“…This compound was synthesized by a modification of published procedures (Hecht et al, 1971). In brief, 6-N-isopentenyladenosine (22) was dissolved in 3.0 ml MeOH containing 0.3 g Raney nickel.…”

Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

“…37 Desulfurized DNA adducts were scanned under acidic (0.1 M HCl), neutral (H 2 O), and alkaline (0.1 M NaOH) conditions.…”

Characterization of Thioether‐Linked Protein Adducts of DNA Using a Raney‐Ni‐Mediated Desulfurization Method and Liquid Chromatography‐Electrospray‐Tandem Mass Spectrometry

“…In order to characterize the structure of various thioether-linked DPCs (e.g., Cys adducts), we developed a novel method using a combination of chemical, enzymatic, and LC-ESI tandem mass spectrometric approaches (Chowdhury et al, 2013). We utilized a Raney Ni-mediated reductive desulfurization approach (Hecht et al, 1971) to convert the bulky DNA-ethylene-protein cross-links into small ethyl adducts that can be readily digested to the nucleoside level and characterized by LC-ESI tandem MS and comparison against authentic standards. In this unit we present a detailed protocol of this method to characterize thioether-linked DPCs.…”