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PMID: 11911366 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

RNA (New York, N.Y.) ·Vol. 8 ·No. 2 ·2002-02-00 ·Pages 202-13

Hansen MA, Kirpekar F, Ritterbusch W, Vester B

Abstract

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 (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis 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.

MeSH Terms
Bacillus subtilis/genetics Base Sequence Escherichia coli/genetics Geobacillus stearothermophilus/genetics Haloarcula marismortui/genetics Models, Molecular Molecular Sequence Data Nucleic Acid Conformation Oligoribonucleotides/chemistry RNA Processing, Post-Transcriptional RNA, Archaeal/chemistry,genetics RNA, Bacterial/chemistry,genetics RNA, Ribosomal, 23S/chemistry,genetics Ribonucleases Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Sulfolobus acidocaldarius/genetics Transcription, Genetic
Chemicals
Oligoribonucleotides RNA, Archaeal RNA, Bacterial RNA, Ribosomal, 23S Ribonucleases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Hansen M A
Department of Molecular Biology, University of Copenhagen, Denmark.
Kirpekar F
Ritterbusch W
Vester B
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838202013365
Published
2002-02-00
Pages
202-13
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1370243
Subset
IM
External Links
PubMed source DOI: 10.1017/s1355838202013365 PMC Full Text View journal details
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