The number and position of the pseudouridines of Haloarcula marismortui and 
Deinococcus radiodurans large subunit RNA have been determined by a combination 
of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine 
sequencing by the reverse transcriptase method and by LC/MS/MS. Three 
pseudouridines were found in H. marismortui, located at positions 1956, 1958, 
and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, 
respectively. The three pseudouridines are all in locations found in other 
organisms. Previous reports of a larger number of pseudouridines in this 
organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine 
(m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 
1900, and 2584, the m3Psi site being determined by a novel application of mass 
spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, 
and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The 
pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in 
positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans 
is conserved in eubacteria and a chloroplast but is not found in archaea or 
eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in 
eukaryotes and is not found in eubacteria. All the pseudoridines are near, but 
not exactly at, nucleotides directly involved in various aspects of ribosome 
function. In addition, two D. radiodurans Psi synthases responsible for the four 
Psi were identified.