• RNA / Volume 8 / Issue 02 / February 2002, pp 202-213
  • Copyright © 2002 RNA Society
  • DOI: http://dx.doi.org/ (About DOI), Published online: 13 February 2002

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria

M.A.  HANSEN a1, F.  KIRPEKAR a2, W.  RITTERBUSCH a2p1 and B.  VESTER a1c1
a1 Department of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen K, Denmark
a2 Department of Biochemistry and Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark


Posttranscriptional modifications were mapped in helices 90–92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90–92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2′-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines ([Psi]s). A total of 10 posttranscriptionally methylated nucleotides and 6 [Psi]s were detected in the five organisms. Eight of the methylated nucleotides and one [Psi] have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

(Received August 16 2001)
(Revised October 4 2001)
(Accepted October 31 2001)

Key Words: 2′-O-ribose methylation; A-site; MALDI mass spectrometry; modified ribonucleotides; pseudouridine; ribosomes.

c1 Reprint requests to: B. Vester, Institute of Molecular Biology, Department of Biological Chemistry, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen K. Denmark; e-mail: birtev@mermaid.molbio.ku.dk.
p1 Present address: Department of Quality Assurance, Arla Foods, Sønderupvej 26, 6920 Videbæk, Denmark.