Having shown that the U18 and snR48 C΄/D΄ motifs both direct multi-site 2΄-O-methylation, we were next interested in determining why these motifs have this unusual property. Starting first with U18, analysis of the U18 C΄/D΄ motif from all available yeast sequences ((24); Figure 2A) revealed that the D΄ box consensus (AUGA) is close to the canonical D΄ box sequence (CUGA) while the C΄ motif contains a single nucleotide insertion (AUGAGUGA). Analysis of the structure of the U18 C΄/D΄ motif revealed that the main variation is found in the stem II region (Figure 2B). One possible explanation for the 2΄-O-methylation of two adjacent nucleotides is that the D΄ region contains two distinct, overlapping D΄ boxes (Figure 2B; left structure AUGA and right structure UAUGA) that would result in two alternative structures for stem II (Figure 2B; canonical and alternative models). In the canonical structure, the extra nucleotide in box C΄ (AUGAGUGA) is not base-paired and bulges out, enabling normal stem II interactions between the two boxes. In the alternative structure model, the extra nucleotide base-pairs with the D΄ box and forms part of stem II. We therefore suggest that the two distinct structures of stem II could result in different positioning of the methyltransferase Nop1 in the complex in each case. The alternative modes of Nop1 binding could therefore place adjacent nucleotides into the active site of Nop1, therefore resulting in the modification of two distinct nucleotides.