Ribosomal RNAs contain many modified nucleotides. The functions of these nucleotides are poorly understood and few of them are strongly conserved. The final stem loop in 16S-like rRNAs is an exception in both regards. In both prokaryotes and eukaryotes, the tetranucleotide loop that caps the 3'-terminal stem contains two N6, N6-dimethyladenosine residues. The sequence and pattern of methylation are conserved within the loop, and there is evidence that these methylated nucleotides play an important role in subunit association and the initiation of protein synthesis. Because of the integral role that helix 45 plays in ribosome function, it is important to know what consequences these methylated nucleotides have on its structure.We have solved the solution structure of a 14-nucleotide analog of the terminal stem loop of bacterial 16S rRNA, which contains N2-methylguanosine as well as two N6,N6-dimethyladenosines.The methylation of the 16S rRNA stem loop completely alters its conformation, which would otherwise be a GNRA tetraloop. It is likely that the conformation of this loop is crucial for its function, having implications for its interaction with ribosomal subunits and its role in the initiation of protein synthesis.