Flexibility of the bulge formed between a hairpin ribozyme and deoxy-substrate analogues

The conformation of the bulge formed between the hairpin ribozyme R derived from (-)sTRSV and noncleavable all-deoxy-substrate analogues dS was studied by photoaffinity labelling. The photolabel deoxy-6-thioinosine was inserted in place of residue G+1 or A-1, located immediately 3' and 5' to the cleavage site, respectively. Upon 335 nm irradiation both substrate analogues were linked to ribozyme at multiple sites. Formation of the R-dS complex is absolutely required for the generation of the crosslinks, since they were detected neither in the absence of Mg2+ nor upon using a ds6I containing 14-mer, unable to interact with the ribozyme. The fraction of ribozyme crosslinked at completion of the reaction increased with increasing analogue concentrations, yielding apparent KD values for the R-dS complex in the range of 5 +/- 2 microM. Multiple crosslinks between ribozyme and each one of the substrate analogues provide clear evidence for a large flexibility of the bulge region.