An extra nucleotide in the consensus catalytic core of a viroid hammerhead ribozyme: implications for the design of more efficient ribozymes

Hammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of the trans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10-->C and A10-->G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10-->U substitution induced a 3-4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes.