Knowledge of the sites, structures, and functional roles of posttranscriptional modification in rRNAs is limited, despite steadily accumulating evidence that rRNA plays a direct role in the peptidyl transferase reaction and that modified nucleotides are concentrated at the functional center of the ribosome. Using methods based on mass spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the central loop of domain V, a region of established interaction between 23 S RNA and tRNA. Two segments of RNA were isolated following protection with oligodeoxynucleotides and nuclease digestion: residues 2423-2473 (51-mer) and 2481-2519 (39-mer). Dihydrouridine was located at position 2449, within the RNase T1 hydrolysis product 2448-ADAACAGp-2454, as evidenced by a molecular mass 2 daltons higher than the gene sequence-predicted mass. This nucleoside, which is nearly ubiquitous in tRNA (where it is involved in maintenance of loop structure), is two bases from A-2551, a previously determined site of interaction between 23 S RNA and the CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately mass measured, and its nucleoside constituents were characterized by high performance liquid chromatography-mass spectrometry; there was no evidence of modification at position 2501 as implied by earlier work. Using similar techniques, the modified adenosine at position 2503 was unambiguously determined to be 2-methyladenosine in the fragment 2503-m2A psi Gp-2505.

The aflylic thioether bond of the prenylcysteines of prenylated proteins has been shown to be cleaved by 2-naphthol under alkaline conditions to yield substituted naphthopyrans. These products are readily resolved from interfering materials by HPLC and have a strongly absorbing chromophore. Thus, this reaction is suitable for quantitative analysis of prenyl substituents of proteins, and we have examined a number of tissues for their content of prenylcysteines. These amino acids are present in mammalian tissues at a concentration of 0.36-1.4 nmol/mg of protein, with a ratio of geranylgeranylcysteine to farnesylcysteine in the range of 4 to 10. Prenylcysteines were also found in the cytosolic fraction of two mouse tissues at about one-third the concentration of the whole organ. The level of these modified amino acids was found to be sigicantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect. Again, geranylgeranylcysteine is predominant. Prenylcysteines were absent from Eschenchia coil but present in an archaebacterium. The prenylcysteine content of mmalan tissue is about 1% of that of cholesterol and about equal to that of ubiquinones and dolichols. Calculations indicate that about 0.5% of all proteins are prenylated.Prenylated proteins contain isoprenoid-modified cysteines in the carboxyl-terminal region (1-3). This posttranslational modification has usually been identified by metabolic labeling of proteins of cells in culture with radioisotopic mevalonate. Metabolic labeling is limiting because many organisms-e.g., yeasts and plants-do not incorporate this precursor readily. In addition, this technique may be impractical, since large doses of isotope would be required for studies with animals. The specific isoprenoid involved with this amino acid (4, 5) has been established by protein hydrolysis and isolation of the prenylcysteine (6) or by cleavage of the prenylcysteine with iodomethane (7) or Raney nickel (4, 5) followed by isolation of the cleavage product. Recently, fast atom bombardment mass spectrometry has been reported as a convenient method for qualitative analysis of prenylated peptides (8). Quantitative analysis with these techniques by direct determination of mass would be difficult if not impossible. Consequently, an alternative method for identifying prenyl modifications would be useful. In prenylated proteins the allylic thioether bond of the prenylcysteines is unique and provides an avenue for selective reactions for identification of prenyl groups. A good nucleophile should react preferentially with and cleave the isoprenoid from cysteine. Prenyl groups linked through oxygen or nitrogen would be expected to be unreactive. For example, a model study using radioactive farnesyl pyrophosphate and our naphthoxide conditions showed about 0.1% of a radioactive product. If the reagent possessed a strongly absorbing chromophoric group, then the derivative would provide a means for detection of the product. We have allowed prenylated proteins to react with 2-naphthol and hav...

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.

No abstract

Mass spectrometry-based methods have been used to study post-transcriptional modification in the 1900-1974 nt segment of domain IV in 23S rRNA of Escherichia coli, a region which interacts with domain V in forming the three- dimensional structure of the peptidyl transferase center within the ribosome. A nucleoside constituent of M r 258 (U*)which occurs at position 1915, within the highly modified oligonucleotide sequence 1911-psiAACU*Apsi-1917, was characterized as 3-methylpseudouridine (m3psi). The assignment was confirmed by chemical synthesis of m3psi and comparison with the natural nucleoside by liquid chromatography-mass spectrometry. 3-Methylpseudouridine is previously unknown in nature and is the only known derivative of the common modified nucleoside pseudouridine thus far found in bacterial rRNA.