The hairpin ribozyme substrate binding-domain: A highly constrained D-shaped conformation

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order t o form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crossl inking and structural approaches in conjunction with molecular modeling (co nstraint-satisfaction program MC-SYM), we have investigated the conformatio n of the substrate-binding domain in the context of the active docked riboz yme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked st ate of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-bin ding strand to cleave a circular substrate, and provides essential informat ion concerning the organization of the substrate in the active conformation . The internal geometry of the substrate-binding strand places G8 of the su bstrate-binding strand near the cleavage site, which has allowed us to pred ict the crucial role played by this nucleotide in the reaction chemistry. ( C) 2001 Academic Press.