Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria
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: firstname.lastname@example.org.
p1 Present address: Department of Quality Assurance, Arla Foods, Sønderupvej 26, 6920 Videbæk, Denmark.