Strategy of mutagenesis on stem-loop 6. Initial design of mutations focused on synonymous codon positions. However, in all but a few instances, this was limited due to the distinctive sequence organization of the apical loop and base paired stem. The first and third codons of the conserved MPN tripeptide (loop region) are limited in respect of variation; M could not be changed and N has two possible silent variations both of which are outside the apical loop ( Figure 3) . Consequently, when mutating the terminal loop sequence UGCCAAAU, silent substitutions could only be introduced into the P codon. Similar difficulties were encountered with mutagenesis of the stem, in which the vast majority of possible synonymous and non-synonymous mutations resulted in no significant conformational changes. The MFold-simulated folding of numerous SL6-mutants revealed a high level of evolutionary 'protection' of SL6 against spontaneous single mutations (not shown) and provides additional evidence for the maintenance of SL6 functionality.