Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which s ites of modification were previously reported, was examined using mass spec trometry. A census of all modified residues was taken by Liquid chromatogra phy/electrospray ionization-mass spectrometry analysis of a total nucleosid e digest of the rRNA. Following rRNA hydrolysis by RNase T-1, accurate mole cular mass values of oligonucleotide products were measured using liquid ch romatography/electrospray ionization-mass spectrometry and compared with va lues predicted from the corresponding gene sequence. Three modified nucleos ides, distributed over four con served sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N-6-m ethyladenosine-1501, and N-6,N-6-dimethyladenosine-1518 and -1519 tall Esch erichia coli numbering). Nueleoside 3-(3-amino-3-carboxypropyl)uridine, pre viously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N-6-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous loc ation in eukaryotic small subunit rRNA but not in bacteria. H, volcanii sma ll subunit rRNA appears to reflect the phenotypically low modification leve l in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rR NA shown to lack pseudouridine.