Influence of Phylogeny on Posttranscriptional Modification of rRNA in Thermophilic Prokaryotes:  The Complete Modification Map of 16S rRNA of <i>Thermus</i> <i>thermophilus</i><sup>†</sup>

Posttranscriptional modification in RNA generally serves to fine-tune and regulate RNA structure and, in many cases, is relatively conserved and phylogenetically distinct. We report the complete modification map for SSU rRNA from <i>Thermus</i> <i>thermophilus</i>, determined primarily by HPLC/electrospray ionization MS-based methods. <i>Thermus</i> modification levels are significantly lower, and structures at the nucleoside level are very different from those of the archaeal thermophile <i>Sulfolobus</i> <i>solfataricus</i> growing in the same temperature range [Noon, K. R., et al. (1998) <i>J. Bacteriol. 180</i>, 2883−2888]. The <i>Thermus</i> modification map is unexpectedly similar to that of <i>Escherichia</i> <i>coli</i> (11 modified sites), with which it shares identity in 8 of the 14 modifications. Unlike the heavily methylated <i>Sulfolobus </i>SSU RNA, <i>Thermus</i> contains a single ribose-methylated residue, <i>N</i><sup>4</sup>,2‘-<i>O</i>-dimethylcytidine-1402, suggesting that O-2‘-ribose methylation in this bacterial thermophile plays a reduced role in thermostabilization compared with the thermophilic archaea. Adjacent pseudouridine residues were found in the single-stranded 3‘ tail of <i>Thermus</i> 16S rRNA at residues 1540 and 1541 (<i>E</i>. <i>coli</i> numbering) in the anti-Shine−Dalgarno mRNA binding sequence. The present results provide an example of the potential of LC/MS for extensive modification mapping in large RNAs.