Folding of DNA substrate-hairpin ribozyme domains: use of deoxy 4-thiouridine as an intrinsic photolabel

摘要

Hairpin ribozymes derived from (?)sTRSV RNA exhibit substantial cleavage activity when wobble GU base pairs are Introduced in place of the AU pairs normally Involved In helices I and II between substrate and ribozyme. This finding prompted us to synthesize by In vitro transcription a new hairpin ribozyme, active against a 14-mer substrate derived from a conserved HIV sequence. Interactions of the canonical and antl-HIV hairpin ribozymes with non cleavable DNA substrate analogues containing the photoaffinity probe deoxy-4-thiouridine (ds4U) at a single site were investigated. Upon near-UV light Irradiation (365 nm), all these substrate analogues were covalently attached to ribozyme via single or multiple crosslinks. In contrast, no crosslinks were detected using either a DNA substrate analogue lacking ds4U or a ds4U containing ollgomer unrelated to the substrate sequence. As expected, if the dissociation constant Is In the range of 5–15 μM, the yield of crossllnked ribozyme Increased markedly with increasing the substrate analogue concentration. The ribozyme residues Involved In the crosslinks were determined by RNA sequencing. The pattern of crosslinks obtained with the two ribozyme systems provides additional evidence in support of the consensus secondary structure proposed for the hairpin domain. Minor alternative conformations were detected in the case of the (?)sTRSV system.