The Archaeon Haloarcula marismortui has Few Modifications in the Central Parts of its 23 S Ribosomal RNA

Post-transcriptional modifications were mapped in domains II, IV and V of 23 S RNA from the archaeon Haloarcula marismortui. The RNA was investigated by two primer extension techniques using reverse transcriptase and three mass spectrometry techniques. One primer extension technique utilized decreasing concentrations of deoxynucleotide triphosphates to detect 2′-O-ribose methylations and other polymerase blocking modifications. In the other, the rRNA was chemically modified, followed by mild alkaline hydrolysis to map pseudo-uridine groups (Ψs). RNA fragments for mass spectrometry were isolated from 23 S rRNA by site-directed RNase H or mung bean nuclease digestion followed by gel purification. Modified RNase digestion fragments were identified with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the modifications were further studied by tandem MS. Ψs suggested by the primer extension technique were verified by specific cyanoethylation and mass spectrometric detection. A total of only five post-transcriptionally methylated nucleotides and three Ψs were detected in the three 23 S rRNA domains. One of the methylated nucleotides has not been reported while a dispute about the number of Ψs is solved. The limited number of modified nucleotides suggests that H. marismortui does not have special needs for extensive rRNA modifications. We have performed detailed investigations on the three-dimensional location and molecular interactions of the modified nucleotides by computer analysis. Our results show that all the modified positions are at regions with RNA–RNA contacts and all except one are at the surface of the subunit and in functionally important regions.