No abstract

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3'- and 5'- terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates. Algorithms for sequence derivation are embodied in a computer program that requires < 2s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direct sequencing of components of mixtures is shown using an unresolved fraction of unknown oligonucleotides from ribosomal RNA.

Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimafly in the temperature range of 56 (Thermoplasma acidophilum) to 105°C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermnotoga narilima (80WC). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.Posttranscriptional processing of tRNA produces a variety of structurally modified nucleosides, some of which have been shown to be associated with a range of biological functions, including maintenance of translational fidelity and efficiency, codon usage, tRNA-protein interactions, and adaptation to cellular stress (9). More than 75 different nucleotides are presently known in tRNA from all sources, with modifications occurring mostly in the base and less commonly by methylation at 0-2' in ribose. Both the chemical nature and sequence locations of individual modifications are highly selective (for reviews, see references 9, 19, and 32), with numerous distinct differences exhibited among the three primary phylogenetic domains, Archaea, Bacteria, and Eucarya (formerly termed archaebacteria, eubacteria, and eukaryotes, respectively [53]). Knowledge of tRNA modification in thermophiles is important as an initial step in understanding structure-stability relationships in the nucleic acids of these remarkable organisms, which grow optimally around the boiling point of water (43), and in identifying domain-or kingdom-specific nucleoside modifications which may serve as phylogenetic markers. Additionally, knowledge of the distributions of modified nucleosides will be useful in later sequencing studies, particularly in avoiding misidentifications when structurally new nucleosides are encountered.Among archaeal microorganisms, tRNA from Halobacterium volcanji has been the most extensively studied (18,20),

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.

Posttranscriptional modification in RNA generally serves to fine-tune and regulate RNA structure and, in many cases, is relatively conserved and phylogenetically distinct. We report the complete modification map for SSU rRNA from Thermus thermophilus, determined primarily by HPLC/electrospray ionization MS-based methods. Thermus modification levels are significantly lower, and structures at the nucleoside level are very different from those of the archaeal thermophile Sulfolobus solfataricus growing in the same temperature range [Noon, K. R., et al. (1998) J. Bacteriol. 180, 2883-2888]. The Thermus modification map is unexpectedly similar to that of Escherichia coli (11 modified sites), with which it shares identity in 8 of the 14 modifications. Unlike the heavily methylated Sulfolobus SSU RNA, Thermus contains a single ribose-methylated residue, N(4),2'-O-dimethylcytidine-1402, suggesting that O-2'-ribose methylation in this bacterial thermophile plays a reduced role in thermostabilization compared with the thermophilic archaea. Adjacent pseudouridine residues were found in the single-stranded 3' tail of Thermus 16S rRNA at residues 1540 and 1541 (E. coli numbering) in the anti-Shine-Dalgarno mRNA binding sequence. The present results provide an example of the potential of LC/MS for extensive modification mapping in large RNAs.