Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Abstract

Yeast tRNA Phe, enriched in carbon-13 specifically at the naturally occurring methyl groups, has been produced through biosynthesis, then purified, and analyzed. Transfer RNA Phe was purified from the [13C]methyl-enriched, unfractionated tRNA that had been extracted from a methionine auxotroph of Saccharomyces cerevisiae [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. H., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408]. The yeast had been grown in minimal medium supplemented with [13C]methylmethionine. Transfer RNA Phe purity and the full extent of nucleoside modification were confirmed by high-performance liquid chromatography of constituent nucleosides with simultaneous UV spectral identification and quantitation. Mass spectometry of [13C]methyl-enriched nucleosides and NMR of the tRNA indicated an enrichment of at least 70 atom %. Twelve resolved and prominent carbon-13 NMR signals from the tRNA were seen between 10 and 60 ppm. These have been assigned to 13 of the 14 naturally occurring methyl groups. However, the partially resolved signals assigned to the two 5-methylcytidines could not be assigned to their specific nucleoside positions of either 40 or 49 in the molecule. In addition, the partially resolved signals of the two methyl esters of wybutosine could not be distinguished. The methyl group found not to be enriched with 13C is bound to the ring carbon in the hypermodified nucleoside wybutosine (Y). A 13th enriched signal downfield (120.9 ppm) has been assigned to one of the two carbons added to guanosine to form the third ring in the biosynthesis of Y. The 13C enrichment of this ring carbon demonstrates its origin from the methionine methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal Structure of the Radical SAM Enzyme Catalyzing Tricyclic Modified Base Formation in tRNA

“…In the tricyclic base formation, the two carbon atoms (C6 and the 6-substituting methyl group) (Figure 1(b)) should be newly introduced. These carbon atoms were reportedly not derived from the methyl group of SAM, 23 and TYW1 may utilize an unidentified substrate X containing a methyl group. Therefore, the 5′-deoxyadenosyl radical may abstract a hydrogen atom from the methyl group of m 1 G37 or from this unidentified substrate X.…”

Naturally-occurring Modification Restricts the Anticodon Domain Conformational Space of tRNA Phe

“…24 It is the first step in the pathway to the hypermodified, tricyclic wybutosine-37 (yW 37 ) in eucaryotic tRNA Phe (Figure 1). 25 In rabbit reticulocyte tRNA Phe the subsequent modification of m 1 G 37 to yW 37 further helps to maintain the reading frame. 26 We have previously demonstrated that incorporation of m 1 G 37 contributed to codon recognition on the ribosome 27 for both the ASL Phe and a DNA analogue.…”

Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs

“…NMR studies of 13 C-enriched tRNA Phe implicated the methyl group of methionine as a source for carbon-10 (refer to Fig. 1 for numbering), and for the side chain ester and N 3 -methyl moieties (35). Conflicting evidence obscures understanding the origin of the 3-amino-3-carboxypropyl side chain (36,37).…”