To determine if these sRNAs might function as guides for ribose methylation in ribosomal RNA in a manner similar to eukaryotic C/D box snoRNAs, the sRNAs were examined for potential guide sequences. Regions complementary to rRNA and adjacent to the D or D' boxes were identified for more than half of the sRNAs. Using the D/D' box plus 5 nt rule, we predicted the locations of potential ribose methyl modifications in rRNA and experimentally tested for these using the dNTP concentration-dependent primer extension assay [Maden et al., 1995,Kiss-Laszlo et al., 1996]. In this assay, characteristic ribose 2'-O-methyl pauses are displayed in the reverse transcriptase reactions at low- but not at high-dNTP concentrations. Using both S. acidocaldarius and S. solfataricus total RNAs as template, we were able to identify pause sites at eight predicted methylation sites (Table 5.1). Examples of four such pause sites are shown in Figure 5.3. Gene disruption systems for S. acidocaldarius and most other archaea are currently not available; consequently we were not able to demonstrate the loss of predicted rRNA methylation sites upon disruption of sRNA genes.