The complex between the hairpin ribozyme and its substrate consists of
two domains that must interact in order to form a catalytic complex,
yet experimental evidence concerning the points of interaction between
the two domains has been lacking. Here, we report the use of hydroxyl
radical footprinting to define the interface between the two domains.
Cations that support very efficient ribozyme catalysis (magnesium and
cobalt(III) hexammine) lead to the formation of a docked complex that
features several regions of protection, indicating a solvent-inaccess
ible core within the tertiary structure of the complex. Cations that a
re suboptimal in cleavage reactions do not produce complexes with regi
ons of reduced solvent accessibility. Nucleotides encompassing the sub
strate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected,
suggesting their internalization into the catalytic core. Four distinc
t segments of the ribozyme are protected, including G11-A14, C25-C27,
A38, and U42-A43. Protection of these sites is eliminated when g+1, an
essential base at the cleavage site, is replaced by A. In addition, m
utations which are known to decrease the fraction of docked complexes
decrease or eliminate formation of a solvent-inaccessible core. Taken
together, these observations demonstrate that we have identified the c
atalytic core of the active hairpin ribozyme-substrate complex.