Post-transcriptionally modified nucleosides are constituents of transfer RNA (tRNA) that are known to influence tertiary structure, stability and coding properties. Modifications in unfractionated tRNA from the phylogenetically unique archaeal methanogen Methanopyrus kandleri (optimal growth temperature 98°C) were studied using liquid chromatography-mass spectrometry to establish the extent to which they might differ from those of other methanogens. The exceptionally diverse population of nucleosides included four new nucleosides of unknown structure, and one that was characterized as N 6 -acetyladenosine, a new RNA constituent. The nucleoside modification pattern in M. kandleri tRNA is notably different from that of other archaeal methanogens, and is closer to that of the thermophilic crenarchaeota.
The tricyclic wye nucleoside family of eight known members constitutes one of the most complex and interesting series of posttranscriptionally modified nucleosides in transfer RNA. The principal reaction paths represented in collision-induced dissociation mass spectra of wye bases and their analogs have been studied in order to determine those structural features that can be readily established by mass spectrometry. The main routes of fragmentation are determined by the presence vs. absence of an amino acid side chain at C-7 (1H-imidazo[1,2-a]purine nomenclature). The common methionine-related side chain is cleaved at two points, providing a ready means of establishing the presence and net level of side chain modification. For those molecules without a side chain, the initial reaction steps are characteristically controlled by the presence vs. absence of methyl at N-4, allowing determination of the methylation status of that site. In the latter case initial opening of the central (pyrimidine) ring, in analogy to the dissociation behavior of guanine, causes loss of identity of C-6/C-7 so that placement of a single methyl at either site is not possible. Subsequent complex reaction paths follow, which include loss of CO and sequential loss of two molecules of HCN.
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