Abstract
Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously 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 location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.
Highlights
LC/ESI-MS of Oligonucleotides from RNase T1 Digests of H. volcanii 16 S rRNA—The mass spectrometer and liquid chromatograph are described in the preceding section; a Z-spray interface was available for these studies
The posttranscriptional modification status of H. volcanii rRNA was examined using a combination of LC/MS-based methods [33] involving analysis of mixtures of nucleosides produced by total enzymatic hydrolysis [31] and of oligonucleotides from RNase T1 digestion
The latter analysis, carried out directly on the total rRNA digest, provides accurate molecular mass values for oligonucleotide products, which can in turn be converted to base compositions [34] and correlated with specific oligonucleotide sequences in the rRNA through comparison with the corresponding gene sequence [35]
Summary
Isolation and Enzymatic Hydrolysis of rRNA—H. volcanii (ATCC 29605) was grown as reported earlier [28]. 30 S ribosomal subunits were prepared [29], and the 16 S rRNA was isolated by extraction with phenol and chloroform [29]. LC/ESI-MS of Nucleosides from Total Digestion of H. volcanii 16 S rRNA—A Quattro II mass spectrometer with MassLynx version 3.1 data system (Micromass, Beverly, MA) interfaced to an HP 1090 liquid chromatograph with diode array detector (Hewlett-Packard, Palo Alto, CA) was used for all LC/MS studies. LC/ESI-MS of Oligonucleotides from RNase T1 Digests of H. volcanii 16 S rRNA—The mass spectrometer and liquid chromatograph are described in the preceding section; a Z-spray interface was available for these studies. Data were acquired in continuum mode over the mass range 100 –350 in 0.4 s (with a 0.1-s interscan delay, for a cycle time of 0.5 s)
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