Alignment of the two domains of the hairpin ribozyme-substrate complex defined by interdomain photoaffinity crosslinking

The hairpin ribozyme-substrate complex contains two independently folding d omains that interact with one another to form a catalytic complex. However, little is known about the key structural elements involved in these tertia ry interactions. Here, we report the use of a photochemical crosslinking me thod to investigate the relative proximity and orientation of the two domai ns of the hairpin ribozyme. This method allows the incorporation of a photo chemical azidophenacyl group at specified positions within synthetic oligor ibonucleotides. Photocrosslinking was performed following the assembly of f our RNA oligonucleotides into active ribozyme-substrate complexes. Two phot oagent attachment sites in the substrate binding strand within domain A (be tween positions A7-G8 and A10-G11) and three in the 5' strand of domain B ( A20-G21, A22-A23 and A24-C25) were studied. Several crosslinks between the substrate binding strand and the 5' segment of domain B were detected. All of the photoagent-specific crosslinked species were dependent upon proper a ssembly and folding of the ribozyme-substrate complex. In addition, a subst rate base mutation (G(+1) to A(+1)) that prevents the docking of the two do mains, blocks the crosslink formation. Four interdomain crosslinks (A7-G8/C 25-A26 (two species); A10-G11/A22 and A24-C25/C12-G13) have been shown to r etain catalytic activity. Taken together, these results indicate that the c haracterized crosslinks provide important information concerning the alignm ent of the two domains and accurately reflect the active docked conformatio n of the molecule. (C) 1999 Academic Press.